Pyrrolobenzodiazepine-antibody conjugates

ABSTRACT

Conjugates of an antibody that binds to CD19 with PBD dimers.

The present invention relates to pyrrolobenzodiazepines (PBDs) having alabile C2 or N10 protecting group in the form of a linker to anantibody.

BACKGROUND TO THE INVENTION

Pyrrolobenzodiazepines

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. andThurston, D. E., Chem. Rev. 2011 111 (4), 2815-2864). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position which is the electrophilic centreresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11a position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res.,19, 230-237 (1986)). Their ability to form an adduct in the minorgroove, enables them to interfere with DNA processing, hence their useas antitumour agents.

A particularly advantageous pyrrolobenzodiazepine compound is describedby Gregson et al. (Chem. Commun. 1999, 797-798) as compound 1, and byGregson et al. (J. Med. Chem. 2001, 44, 1161-1174) as compound 4a. Thiscompound, also known as SG2000, is shown below:

WO 2007/085930 describes the preparation of dimer PBD compounds havinglinker groups for connection to a cell binding agent, such as anantibody. The linker is present in the bridge linking the monomer PBDunits of the dimer.

The present inventors have described dimer PBD compounds having linkergroups for connection to a cell binding agent, such as an antibody, inWO 2011/130613 and WO 2011/130616. The linker in these compounds isattached to the PBD core via the C2 position, and are generally cleavedby action of an enzyme on the linker group. In WO 2011/130598, thelinker in these compounds is attached to one of the available N10positions on the PBD core, and are generally cleaved by action of anenzyme on the linker group.

Antibody-Drug Conjugates

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders (Carter, P.(2006) Nature Reviews Immunology 6:343-357). The use of antibody-drugconjugates (ADC), i.e. immunoconjugates, for the local delivery ofcytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumorcells in the treatment of cancer, targets delivery of the drug moiety totumors, and intracellular accumulation therein, whereas systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells (Xie et al (2006)Expert. Opin. Biol. Ther. 6(3):281-291; Kovtun et al (2006) Cancer Res.66(6):3214-3121; Law et al (2006) Cancer Res. 66(4):2328-2337; Wu et al(2005) Nature Biotech. 23(9):1137-1145; Lambert J. (2005) Current Opin.in Pharmacol. 5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents15(9):1087-1103; Payne, G. (2003) Cancer Cell 3:207-212; Trail et al(2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos(1999) Anticancer Research 19:605-614).

Maximal efficacy with minimal toxicity is sought thereby. Efforts todesign and refine ADC have focused on the selectivity of monoclonalantibodies (mAbs) as well as drug mechanism of action, drug-linking,drug/antibody ratio (loading), and drug-releasing properties (Junutula,et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485;WO2009/052249; McDonagh (2006) Protein Eng. Design & Sel. 19(7):299-307; Doronina et al (2006) Bioconj. Chem. 17:114-124; Erickson et al(2006) Cancer Res. 66(8):1-8; Sanderson et al (2005) Clin. Cancer Res.11:843-852; Jeffrey et al (2005) J. Med. Chem. 48:1344-1358; Hamblett etal (2004) Clin. Cancer Res. 10:7063-7070). Drug moieties may imparttheir cytotoxic and cytostatic effects by mechanisms including tubulinbinding, DNA binding, proteasome and/or topoisomerase inhibition. Somecytotoxic drugs tend to be inactive or less active when conjugated tolarge antibodies or protein receptor ligands.

The present inventors have developed particular PBD dimer antibodyconjugates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the in vitro efficacy of a conjugate of the inventionagainst Daudi, Ramos and WSU-DLCL2 cells.

FIG. 2 shows the in vitro efficacy of another conjugate of the inventionagainst Daudi, Ramos and WSU-DLCL2 cells.

FIG. 3 shows the in vitro efficacy of another conjugate of the inventionagainst Daudi, Ramos and WSU-DLCL2 cells.

FIG. 4 shows the anti-tumour activity of conjugates of the invention andconjugates not of the invention in a Ramos xenograft model.

DISCLOSURE OF THE INVENTION

A first aspect of the present invention comprises a conjugate of formulaL-(D^(L))_(p), where D^(L) is of formula I or II:

wherein:L is an antibody (Ab) as defined below;when there is a double bond present between C2′ and C3′, R¹² is selectedfrom the group consisting of:(ia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;(ib) C₁₋₅ saturated aliphatic alkyl;(ic) C₃₋₆ saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;when there is a single bond present between C2′ and C3′,R¹² is

R^(26a) and R^(26b) are independently selected from H, F, C₁₋₄ saturatedalkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups are optionallysubstituted by a group selected from C₁₋₄ alkyl amido and C₁₋₄ alkylester; or, when one of R^(26a) and R^(26b) is H, the other is selectedfrom nitrile and a C₁₋₄ alkyl ester;R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂,NHR, NRR′, nitro, Me₃Sn and halo;where R and R′ are independently selected from optionally substitutedC₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups;R⁷ is selected from H, R, OH, OR, SH, SR, NH₂, NHR, NHRR′, nitro, Me₃Snand halo;R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one ormore heteroatoms, e.g. O, S, NR^(N2) (where R^(N2) is H or C₁₋₄ alkyl),and/or aromatic rings, e.g. benzene or pyridine;Y and Y′ are selected from O, S, or NH;

R^(6′), R^(7′), R^(9′) are selected from the same groups as R⁶, R⁷ andR⁹ respectively;

[Formula I]

R^(L1′) is a linker for connection to the antibody (Ab);

R^(11a) is selected from OH, OR^(A), where R^(A) is C₁₋₄ alkyl, andSO_(z)M, where z is 2 or 3 and M is a monovalent pharmaceuticallyacceptable cation;

R²⁰ and R²¹ either together form a double bond between the nitrogen andcarbon atoms to which they are bound or;

R²⁰ is selected from H and R^(C), where R^(C) is a capping group;

R²¹ is selected from OH, OR^(A) and SO_(z)M;

when there is a double bond present between C2 and C3, R² is selectedfrom the group consisting of:

(ia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(ib) C₁₋₅ saturated aliphatic alkyl;

(ic) C₃₋₆ saturated cycloalkyl;

wherein each of R¹¹, R¹² and R¹³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5;

wherein one of R^(15a) and R^(15b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R¹⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;when there is a single bond present between C2 and C3,R² is

where R^(16a) and R^(16b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(16a) and R^(16b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester;[Formula II]R²² is of formula IIIa, formula IIIb or formula IIIc:

where A is a C₅₋₇ aryl group, and either(i) Q¹ is a single bond, and Q² is selected from a single bond and—Z—(CH₂)_(n)—, where Z is selected from a single bond, O, S and NH and nis from 1 to 3; or(ii) Q¹ is —CH═CH—, and Q² is a single bond;

where;R^(C1), R^(C2) and R^(C3) are independently selected from H andunsubstituted C₁₋₂ alkyl;

where Q is selected from O—R^(L2′), S—R^(L2′) and NR^(N)—R^(L2′), andR^(N) is selected from H, methyl and ethylX is selected from the group comprising: O—R^(L2′), S—R^(L2′),CO₂—R^(L2′), CO—R^(L2′), NH—C(═O)—R^(L2′), NHNH—R^(L2′), CONHNH—R^(L2′),

NR^(N)R^(L2′), wherein R^(N) is selected from the group comprising H andC₁₋₄ alkyl;R^(L2′) is a linker for connection to the antibody (Ab);R¹⁰ and R¹¹ either together form a double bond between the nitrogen andcarbon atoms to which they are bound or;R¹⁰ is H and R¹¹ is selected from OH, OR^(A) and SO_(z)M;R³⁰ and R³¹ either together form a double bond between the nitrogen andcarbon atoms to which they are bound or;R³⁰ is H and R³¹ is selected from OH, OR^(A) and SO_(z)M.

In some embodiments, the conjugate is not:

In other embodiments, it may be preferred that the conjugate is selectedfrom a conjugate of formula ConjA, ConjB, ConjC, ConjD and ConjE.

The subscript p in the formula I is an integer of from 1 to 20.Accordingly, the Conjugates comprise an antibody (Ab) as defined belowcovalently linked to at least one Drug unit by a Linker unit. The Ligandunit, described more fully below, is a targeting agent that binds to atarget moiety. Accordingly, the present invention also provides methodsfor the treatment of, for example, various cancers and autoimmunedisease. The drug loading is represented by p, the number of drugmolecules per antibody. Drug loading may range from 1 to 20 Drug units(D^(L)) per antibody. For compositions, p represents the average drugloading of the Conjugates in the composition, and p ranges from 1 to 20.

A second aspect of the invention provides a method of making a conjugateaccording to the first aspect of the invention comprising conjugating acompound of formula I^(L) or II^(L):

to the antibody (Ab) as defined below, wherein:R^(L1) is a linker suitable for conjugation to the antibody (Ab);R^(22L) is of formula IIIa^(L), formula IIIb^(L) or formula IIIc^(L):

where Q^(L) is selected from O—R^(L2), S—R^(L2) and NR^(N)—R^(L2), andR^(N) is selected from H, methyl and ethylX^(L) is selected from the group comprising: O—R^(L2), S—R^(L2),CO₂—R^(L2), CO—R^(L2), N═C═O—R^(L2), NHNH—R^(L2), CONHNH—R^(L2),

NR^(N)R^(L), wherein R^(N) is selected from the group comprising H andC₁₋₄ alkyl;R^(L2) is a linker suitable for conjugation to the antibody (Ab);and all the remaining groups are as defined in the first aspect.

Thus it may be preferred in the second aspect, that the inventionprovides a method of making a conjugate selected from the groupconsisting of ConjA, ConjB, ConjC, ConjD and ConjE comprisingconjugating a compound which is selected respectively from

with an antibody as defined below.

WO 2011/130615 discloses compound 26:

which is the parent compound of A. Compound A comprises the PBD with alinker for attachment to a cell binding agent. The cell binding agentprovides a number of ethylene glycol moieties to provide solubilitywhich is useful in the synthesis of conjugates.

WO 2010/043380 and WO 2011/130613 disclose compound 30:

WO 2011/130613 also discloses compound 51:

Compound B differs from compound 30 by only having a (CH₂)₃ tetherbetween the PBD moieties, instead of a (CH₂)₅ tether, which reduces thelipophilicity of the released PBD dimer. The linking group is attachedto the C2-phenyl group in the para rather than meta position.

WO 2011/130613 discloses compound 9351:

Compound C differs from this in two respects. The cell binding agentprovides an increased number of ethylene glycol moieties to providesolubility which is useful in the synthesis of conjugates, and thephenyl substituent provide two rather than one oxygen atom, which alsoaids solubility. Compound C's structure may also mean it binds morestrongly in the minor groove.

Compounds A, B and C have two sp² centres in each C-ring, which mayallow for stronger binding in the minor groove of DNA, than forcompounds with only one sp² centre in each C-ring.

WO 2011/130598 discloses compound 80:

Compound D differs from this by comprising an iodoacetamide group forlinking to the cell binding agent. This group may offer advantages overcompound 80 with regards to its stability when bound to a cell bindingagent (see below). The malemide group in compound 80 can undergo aretro-Michael reaction, becoming unconjugated from the cell bindingagent, and thus vulnerable to scavenging by other thiol containingbiological molecules, such as albumin and glutathione. Suchunconjugation cannot occur with compound A. Also, the iodoacetamidegroup may avoid other unwanted side reactions.

Compound E differs from previously disclosed PBD dimers with a druglinker having a C2-3 endo-double bond, by having a smaller, lesslipophilic C2 substituent, e.g. 4F-phenyl, propylene. As such, theconjugates of compound B (see below) are less likely to aggregate oncesynthesised. Such aggregation of conjugates can be measured by Sizeexclusion chromatography (SEC).

Both compound D and E have two sp² centres in each C-ring, which mayallow for stronger binding in the minor groove of DNA, than forcompounds with only one sp² centre in each C-ring.

The drug linkers disclosed in WO 2010/043880, WO 2011/130613, WO2011/130598 and WO 2011/130616 may be used in the present invention, andare incorporated herein by reference. The drug linkers described hereinmay be synthesised as described in these disclosures.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is suitable for use in providing a PBD compound toa preferred site in a subject. The conjugate allows the release of anactive PBD compound that does not retain any part of the linker. Thereis no stub present that could affect the reactivity of the PBD compound.Thus ConjA would release the compound RelA:

ConjB would release the compound RelB:

ConjC would release the compound RelC:

ConjD would release the compound RelD:

and ConjE would release the compound RelE:

The specified link between the PBD dimer and the antibody, in thepresent invention is preferably stable extracellularly. Before transportor delivery into a cell, the antibody-drug conjugate (ADC) is preferablystable and remains intact, i.e. the antibody remains linked to the drugmoiety. The linkers are stable outside the target cell and may becleaved at some efficacious rate inside the cell. An effective linkerwill: (i) maintain the specific binding properties of the antibody; (ii)allow intracellular delivery of the conjugate or drug moiety; (iii)remain stable and intact, i.e. not cleaved, until the conjugate has beendelivered or transported to its targeted site; and (iv) maintain acytotoxic, cell-killing effect or a cytostatic effect of the PBD drugmoiety. Stability of the ADC may be measured by standard analyticaltechniques such as mass spectroscopy, HPLC, and the separation/analysistechnique LC/MS.

Delivery of the compounds of formulae RelA, RelB, RelC, RelD or RelE isachieved at the desired activation site of the conjugates of formulaeConjA, ConjB, ConjC, ConjD or ConjE by the action of an enzyme, such ascathepsin, on the linking group, and in particular on the valine-alaninedipeptide moiety.

Antibody

In one aspect the antibody is an antibody that binds to CD19, theantibody comprising a VH domain having the sequence according to any oneof SEQ ID NOs. 1, 2, 3, 4, 5 or 6.

The antibody may further comprise a VL domain. In some embodiments theantibody further comprises a VL domain having the sequence according toany one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12.

In some embodiments the antibody comprises a VH domain paired with a VLdomain, the VH and VL domains having sequences selected from the groupconsisting of:

SEQ ID NO. 1 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 2 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 3 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 4 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 5 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;and

SEQ ID NO. 6 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12.For example SEQ ID NO. 1 paired with SEQ ID NO. 7, SEQ ID NO. 2 pairedwith SEQ ID NO. 8, SEQ ID NO. 3 paired with SEQ ID NO. 9, SEQ ID NO. 4paired with SEQ ID NO. 10, SEQ ID NO. 5 paired with SEQ ID NO. 11 or SEQID NO. 6 paired with SEQ ID NO. 12.

The VH and VL domain(s) may pair so as to form an antibody antigenbinding site that binds CD19.

In some embodiments the antibody is an intact antibody comprising a VHdomain paired with a VL domain, the VH and VL domains having sequencesselected from the group consisting of:

SEQ ID NO. 1 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 2 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 3 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 4 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;

SEQ ID NO. 5 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12;and

SEQ ID NO. 6 paired with any one of SEQ ID NOs. 7, 8, 9, 10, 11 or 12.For example SEQ ID NO. 1 paired with SEQ ID NO. 7, SEQ ID NO. 2 pairedwith SEQ ID NO. 8, SEQ ID NO. 3 paired with SEQ ID NO. 9, SEQ ID NO. 4paired with SEQ ID NO. 10, SEQ ID NO. 5 paired with SEQ ID NO. 11 or SEQID NO. 6 paired with SEQ ID NO. 12.

In some embodiments, the antibody competes with the antibody secreted byhybridoma ATCC accession No. HB-305 for binding to CD19. In oneembodiment the antibody binds CD19 with an association constant (K_(a))no less than 2, 5 or 10-fold less than the antibody secreted by thehybridoma.

In aspect the antibody is an antibody as described herein which has beenmodified (or further modified) as described below. In some embodimentsthe antibody is a humanised, deimmunised or resurfaced version of anantibody disclosed herein.

Terminology

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), intact antibodies and antibody fragments, so long as theyexhibit the desired biological activity, for example, the ability tobind CD19 (Miller et al (2003) Jour. of Immunology 170:4854-4861).Antibodies may be murine, human, humanized, chimeric, or derived fromother species. An antibody is a protein generated by the immune systemthat is capable of recognizing and binding to a specific antigen.(Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology,5th Ed., Garland Publishing, New York). A target antigen generally hasnumerous binding sites, also called epitopes, recognized by CDRs onmultiple antibodies. Each antibody that specifically binds to adifferent epitope has a different structure. Thus, one antigen may havemore than one corresponding antibody. An antibody includes a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin can be of anytype (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass, or allotype (e.g. human G1m1, G1 m2,G1 m3, non-G1m1 [that, is any allotype other than G1m1], G1m17, G2m23,G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6,G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2 and Km3) of immunoglobulinmolecule. The immunoglobulins can be derived from any species, includinghuman, murine, or rabbit origin.

As used herein, “binds CD19” is used to mean the antibody binds CD19with a higher affinity than a non-specific partner such as Bovine SerumAlbumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1GI:3336842, record update date: Jan. 7, 2011 02:30 PM). In someembodiments the antibody binds CD19 with an association constant (K_(a))at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10⁴,10⁵ or 10⁶-fold higher than the antibody's association constant for BSA,when measured at physiological conditions. The antibodies of theinvention can bind CD19 with a high affinity. For example, in someembodiments the antibody can bind CD19 with a K_(D) equal to or lessthan about 10⁻⁶ M, such as 1×10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹², 10⁻¹³ or 10⁻¹⁴

In some embodiments, CD19 polypeptide corresponds to Genbank accessionno. NP_001171569, version no. NP_001171569.1 GI:296010921, record updatedate: Sep. 10, 2012 12:43 AM. In one embodiment, the nucleic acidencoding CD19 polypeptide corresponds to Genbank accession noNM_001178098, version no. NM_001178098.1 GI:296010920, record updatedate: Sep. 10, 2012 12:43 AM.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and scFv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carryinga fully human immunoglobulin system (Lonberg (2008) Curr. Opinion20(4):450-459).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodiesinclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g. OldWorld Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising VL and VH domains, as wellas a light chain constant domain (CL) and heavy chain constant domains,CH1, CH2 and CH3. The constant domains may be native sequence constantdomains (e.g. human native sequence constant domains) or amino acidsequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ϵ, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Modification of Antibodies

The antibodies disclosed herein may be modified. For example, to makethem less immunogenic to a human subject. This may be achieved using anyof a number of techniques familiar to the person skilled in the art.Some of these techniques are described in more detail below.

Humanisation

Techniques to reduce the in vivo immunogenicity of a non-human antibodyor antibody fragment include those termed “humanisation”.

A “humanized antibody” refers to a polypeptide comprising at least aportion of a modified variable region of a human antibody wherein aportion of the variable region, preferably a portion substantially lessthan the intact human variable domain, has been substituted by thecorresponding sequence from a non-human species and wherein the modifiedvariable region is linked to at least another part of another protein,preferably the constant region of a human antibody. The expression“humanized antibodies” includes human antibodies in which one or morecomplementarity determining region (“CDR”) amino acid residues and/orone or more framework region (“FW” or “FR”) amino acid residues aresubstituted by amino acid residues from analogous sites in rodent orother non-human antibodies. The expression “humanized antibody” alsoincludes an immunoglobulin amino acid sequence variant or fragmentthereof that comprises an FR having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Or, looked at another way, a humanized antibody is ahuman antibody that also contains selected sequences from non-human(e.g. murine) antibodies in place of the human sequences. A humanizedantibody can include conservative amino acid substitutions ornon-natural residues from the same or different species that do notsignificantly alter its binding and/or biologic activity. Suchantibodies are chimeric antibodies that contain minimal sequence derivedfrom non-human immunoglobulins.

There are a range of humanisation techniques, including ‘CDR grafting’,‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known as‘veneering’), ‘composite antibodies’, ‘Human String ContentOptimisation’ and framework shuffling.

CDR Grafting

In this technique, the humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient antibody are replaced by residues from aCDR of a non-human species (donor antibody) such as mouse, rat, camel,bovine, goat, or rabbit having the desired properties (in effect, thenon-human CDRs are ‘grafted’ onto the human framework). In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues (this may happen when,for example, a particular FR residue has significant effect on antigenbinding).

Furthermore, humanized antibodies can comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. Thus, in general, a humanized antibody willcomprise all of at least one, and in one aspect two, variable domains,in which all or all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), or that of a human immunoglobulin.

Guided Selection

The method consists of combining the V_(H) or V_(L) domain of a givennon-human antibody specific for a particular epitope with a human V_(H)or V_(L) library and specific human V domains are selected against theantigen of interest. This selected human VH is then combined with a VLlibrary to generate a completely human VH×VL combination. The method isdescribed in Nature Biotechnology (N.Y.) 12, (1994) 899-903.

Composite Antibodies

In this method, two or more segments of amino acid sequence from a humanantibody are combined within the final antibody molecule. They areconstructed by combining multiple human VH and VL sequence segments incombinations which limit or avoid human T cell epitopes in the finalcomposite antibody V regions. Where required, T cell epitopes arelimited or avoided by, exchanging V region segments contributing to orencoding a T cell epitope with alternative segments which avoid T cellepitopes. This method is described in US 2008/0206239 A1.

Deimmunization

This method involves the removal of human (or other second species)T-cell epitopes from the V regions of the therapeutic antibody (or othermolecule). The therapeutic antibodies V-region sequence is analysed forthe presence of MHC class II-binding motifs by, for example, comparisonwith databases of MHC-binding motifs (such as the “motifs” databaseavailable on the website of the Walter+Eliza Hall Institute of MedicalResearch). Alternatively, MHC class II-binding motifs may be identifiedusing computational threading methods such as those devised by Altuviaet al. (J. Mol. Biol. 249 244-250 (1995)); in these methods, consecutiveoverlapping peptides from the V-region sequences are testing for theirbinding energies to MHC class II proteins. This data can then becombined with information on other sequence features which relate tosuccessfully presented peptides, such as amphipathicity, Rothbardmotifs, and cleavage sites for cathepsin B and other processing enzymes.

Once potential second species (e.g. human) T-cell epitopes have beenidentified, they are eliminated by the alteration of one or more aminoacids. The modified amino acids are usually within the T-cell epitopeitself, but may also be adjacent to the epitope in terms of the primaryor secondary structure of the protein (and therefore, may not beadjacent in the primary structure). Most typically, the alteration is byway of substitution but, in some circumstances amino acid addition ordeletion will be more appropriate.

All alterations can be accomplished by recombinant DNA technology, sothat the final molecule may be prepared by expression from a recombinanthost using well established methods such as Site Directed Mutagenesis.However, the use of protein chemistry or any other means of molecularalteration is also possible.

Resurfacinq

This method involves:

-   -   (a) determining the conformational structure of the variable        region of the non-human (e.g. rodent) antibody (or fragment        thereof) by constructing a three-dimensional model of the        non-human antibody variable region;    -   (b) generating sequence alignments using relative accessibility        distributions from x-ray crystallographic structures of a        sufficient number of non-human and human antibody variable        region heavy and light chains to give a set of heavy and light        chain framework positions wherein the alignment positions are        identical in 98% of the sufficient number of non-human antibody        heavy and light chains;    -   (c) defining for the non-human antibody to be humanized, a set        of heavy and light chain surface exposed amino acid residues        using the set of framework positions generated in step (b);    -   (d) identifying from human antibody amino acid sequences a set        of heavy and light chain surface exposed amino acid residues        that is most closely identical to the set of surface exposed        amino acid residues defined in step (c), wherein the heavy and        light chain from the human antibody are or are not naturally        paired;    -   (e) substituting, in the amino acid sequence of the non-human        antibody to be humanized, the set of heavy and light chain        surface exposed amino acid residues defined in step (c) with the        set of heavy and light chain surface exposed amino acid residues        identified in step (d);    -   (f) constructing a three-dimensional model of the variable        region of the non-human antibody resulting from the substituting        specified in step (e);    -   (g) identifying, by comparing the three-dimensional models        constructed in steps (a) and (f), any amino acid residues from        the sets identified in steps (c) or (d), that are within 5        Angstroms of any atom of any residue of the complementarity        determining regions of the non-human antibody to be humanized;        and    -   (h) changing any residues identified in step (g) from the human        to the original non-human amino acid residue to thereby define a        non-human antibody humanizing set of surface exposed amino acid        residues; with the proviso that step (a) need not be conducted        first, but must be conducted prior to step (g).        Superhumanization

The method compares the non-human sequence with the functional humangermline gene repertoire. Those human genes encoding canonicalstructures identical or closely related to the non-human sequences areselected. Those selected human genes with highest homology within theCDRs are chosen as FR donors. Finally, the non-human CDRs are graftedonto these human FRs. This method is described in patent WO 2005/079479A2.

Human String Content Optimization

This method compares the non-human (e.g. mouse) sequence with therepertoire of human germline genes and the differences are scored asHuman String Content (HSC) that quantifies a sequence at the level ofpotential MHC/T-cell epitopes. The target sequence is then humanized bymaximizing its HSC rather than using a global identity measure togenerate multiple diverse humanized variants (described in MolecularImmunology, 44, (2007) 1986-1998).

Framework Shuffling

The CDRs of the non-human antibody are fused in-frame to cDNA poolsencompassing all known heavy and light chain human germline geneframeworks. Humanised antibodies are then selected by e.g. panning ofthe phage displayed antibody library. This is described in Methods 36,43-60 (2005).

Definitions

Pharmaceutically Acceptable Cations

Examples of pharmaceutically acceptable monovalent and divalent cationsare discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977), whichis incorporated herein by reference.

The pharmaceutically acceptable cation may be inorganic or organic.

Examples of pharmaceutically acceptable monovalent inorganic cationsinclude, but are not limited to, alkali metal ions such as Na⁺ and K⁺.Examples of pharmaceutically acceptable divalent inorganic cationsinclude, but are not limited to, alkaline earth cations such as Ca²⁺ andMg²⁺. Examples of pharmaceutically acceptable organic cations include,but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substitutedammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). The term “C₁₋₄ alkyl”as used herein, pertains to a monovalent moiety obtained by removing ahydrogen atom from a carbon atom of a hydrocarbon compound having from 1to 4 carbon atoms, which may be aliphatic or alicyclic, and which may besaturated or unsaturated (e.g. partially unsaturated, fullyunsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl,alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:        cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),        cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),        dimethylcyclopropane (C₅), methylcyclobutane (C₅),        dimethylcyclobutane (C₆), methylcyclopentane (C₆),        dimethylcyclopentane (C₇) and methylcyclohexane (C₇);    -   unsaturated monocyclic hydrocarbon compounds:        cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),        cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene        (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),        methylcyclopentene (C₆), dimethylcyclopentene (C₇) and        methylcyclohexene (C₇); and    -   saturated polycyclic hydrocarbon compounds:        norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);O₃: trioxane (C₆);N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);N₂O₁: oxadiazine (C₆);O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. The term “C₅₋₇ aryl”, as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from an aromatic ring atomof an aromatic compound, which moiety has from 5 to 7 ring atoms and theterm “C₅₋₁₀ aryl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 5 to 10 ring atoms. Preferably,each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, C₅₋₁₀, etc.)denote the number of ring atoms, or range of number of ring atoms,whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl”as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (C₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (C₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene        (O₁), thioxanthene (S₁), oxanthrene (C₂), phenoxathiin (O₁S₁),        phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁),        thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂),        phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇alkyl group.Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetalsubstituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in thecase of a “cyclic” acetal group, R¹ and R², taken together with the twooxygen atoms to which they are attached, and the carbon atoms to whichthey are attached, form a heterocyclic ring having from 4 to 8 ringatoms. Examples of acetal groups include, but are not limited to,—CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groupsinclude, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and Ris a ketal substituent other than hydrogen, for example, a C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably aC₁₋₇ alkyl group. Examples ketal groups include, but are not limited to,—C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂,—C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and Ris a hemiketal substituent other than hydrogen, for example, a C₁₋₇alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include,but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),—C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).Oxo (keto, -one): ═O.Thione (thioketone): ═S.Imino (imine): ═NR, wherein R is an imino substituent, for example,hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably hydrogen or a C₁₋₇ alkyl group. Examples of estergroups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, aC₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl),a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl),or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably aC₁₋₇ alkyl group. Examples of acyl groups include, but are not limitedto, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃(t-butyryl), and —C(═O)Ph (benzoyl, phenone).Carboxy (carboxylic acid): —C(═O)OH.Thiocarboxy (thiocarboxylic acid): —C(═S)SH.Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.Thionocarboxy (thionocarboxylic acid): —C(═S)OH.Imidic acid: —C(═NH)OH.Hydroxamic acid: —C(═NOH)OH.Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent,for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groupsinclude, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃,—OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of ester groups include,but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃,and —OC(═O)OPh.Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case ofa “cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² areindependently amino substituents, as defined for amino groups. Examplesof amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃,—C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R²is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide groups include, but are not limitedto, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may togetherform a cyclic structure, as in, for example, succinimidyl, maleimidyl,and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independentlyamino substituents, as defined for amino groups. Examples ofaminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,—OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.Ureido: —N(R′)CONR²R³ wherein R² and R³ are independently aminosubstituents, as defined for amino groups, and R¹ is a ureidosubstituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ureido groups include, but are not limited to,—NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂,—NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.Guanidino: —NH—C(═NH)NH₂.Tetrazolyl: a five membered aromatic ring having four nitrogen atoms andone carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example,hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groupsinclude, but are not limited to, ═NH, ═NMe, and ═NEt.Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent,for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group,or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples ofamidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂,and —C(═NMe)NMe₂.Nitro: —NO₂.Nitroso: —NO.Azido: —N₃.Cyano (nitrile, carbonitrile): —CN.Isocyano: —NC.Cyanato: —OCN.Isocyanato: —NCO.Thiocyano (thiocyanato): —SCN.Isothiocyano (isothiocyanato): —NCS.Sulfhydryl (thiol, mercapto): —SH.Thioether (sulfide): —SR, wherein R is a thioether substituent, forexample, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group),a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇alkyl group. Examples of C₁₋₇ alkylthio groups include, but are notlimited to, —SCH₃ and —SCH₂CH₃.Disulfide: —SS—R, wherein R is a disulfide substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyldisulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are notlimited to, —SSCH₃ and —SSCH₂CH₃.Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfinesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfine groups include, but are not limited to, —S(═O)CH₃ and—S(═O)CH₂CH₃.Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group, including, for example, afluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfonegroups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl,mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C4F₉(nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph(phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl),4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl),4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfinate groups include, but are not limited to, —S(═O)OCH₃(methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl;ethyl sulfinate).Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonatesubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ofsulfonate groups include, but are not limited to, —S(═O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl;ethyl sulfonate).Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxy groupsinclude, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, forexample, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groupsinclude, but are not limited to, —OS(═O)₂CH₃ (mesylate) and—OS(═O)₂CH₂CH₃ (esylate).Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, butare not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of sulfamyl groups include, but are not limitedto, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃),—S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):—S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, asdefined for amino groups. Examples of sulfonamido groups include, butare not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂,—S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.Sulfamino: —NR'S(═O)₂OH, wherein R¹ is an amino substituent, as definedfor amino groups. Examples of sulfamino groups include, but are notlimited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.Sulfonamino: —NR'S(═O)₂R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfonamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include,but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as definedfor amino groups, and R is a sulfinamino substituent, for example, aC₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group,preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include,but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, forexample, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphino groups include, but are not limited to, —PH₂,—P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.Phospho: —P(═O)₂.Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinylsubstituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not limitedto, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.Phosphonic acid (phosphono): —P(═O)(OH)₂.Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphonate groups include, but are notlimited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and—P(═O)(OPh)₂.Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphatesubstituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclylgroup, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or aC₅₋₂₀ aryl group. Examples of phosphate groups include, but are notlimited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and—OP(═O)(OPh)₂.Phosphorous acid: —OP(OH)₂.Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example,—H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ arylgroup, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group.Examples of phosphite groups include, but are not limited to,—OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.Phosphoramidite: —OP(OR¹)—NR² ₂, where R¹ and R² are phosphoramiditesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramiditegroups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂,—OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.Phosphoramidate: —OP(═O)(OR¹)—NR² ₂, where R¹ and R² are phosphoramidatesubstituents, for example, —H, a (optionally substituted) C₁₋₇ alkylgroup, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H,a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidategroups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂,—OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to abidentate moiety obtained by removing two hydrogen atoms, either bothfrom the same carbon atom, or one from each of two different carbonatoms, of a hydrocarbon compound having from 3 to 12 carbon atoms(unless otherwise specified), which may be aliphatic or alicyclic, andwhich may be saturated, partially unsaturated, or fully unsaturated.Thus, the term “alkylene” includes the sub-classes alkenylene,alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are notlimited to, —(CH₂)_(n)— where n is an integer from 3 to 12, for example,—CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂—(pentylene) and —CH₂CH₂CH₂CH—₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂ alkylene groups include, but arenot limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—,—CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and—CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene, and alkynylene groups) include, but are not limited to,—CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—,—CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—,—CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—CC≡CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂alkenylene and alkynylene groups) include, but are not limited to,—C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C≡CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentylene (e.g.cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g.4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene;3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Carbamate nitrogen protecting group: the term “carbamate nitrogenprotecting group” pertains to a moiety which masks the nitrogen in theimine bond, and these are well known in the art. These groups have thefollowing structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 503 to 549 of Greene, T. W. and Wuts, G. M.,Protective Groups in Organic Synthesis, 3′ Edition, John Wiley & Sons,Inc., 1999, which is incorporated herein by reference.

Hemi-aminal nitrogen protecting group: the term “hemi-aminal nitrogenprotecting group” pertains to a group having the following structure:

wherein R′¹⁰ is R as defined above. A large number of suitable groupsare described on pages 633 to 647 as amide protecting groups of Greene,T. W. and Wuts, G. M., Protective Groups in Organic Synthesis, 3^(rd)Edition, John Wiley & Sons, Inc., 1999, which is incorporated herein byreference.

The groups Carbamate nitrogen protecting group and Hemi-aminal nitrogenprotecting group may be jointly termed a “nitrogen protecting group forsynthesis”.

Conjugates

The present invention provides a conjugate comprising a PBD compoundconnected to the antibody via a Linker Unit.

In one embodiment, the conjugate comprises the antibody connected to aspacer connecting group, the spacer connected to a trigger, the triggerconnected to a self-immolative linker, and the self-immolative linkerconnected to the N10 position of the PBD compound. Such a conjugate isillustrated below:

where Ab is the antibody as defined above and PBD is apyrrolobenzodiazepine compound (D), as described herein. Theillustration shows the portions that correspond to R^(L′), A, L¹ and L²in certain embodiments of the invention. R^(L′) may be either R^(L1′) orR^(L2′). D is D^(L) with R^(L1′) or R^(L2′) removed.

The present invention is suitable for use in providing a PBD compound toa preferred site in a subject. In the preferred embodiments, theconjugate allows the release of an active PBD compound that does notretain any part of the linker. There is no stub present that couldaffect the reactivity of the PBD compound.

The linker attaches the antibody to the PBD drug moiety D throughcovalent bond(s). The linker is a bifunctional or multifunctional moietywhich can be used to link one or more drug moiety (D) and an antibodyunit (Ab) to form antibody-drug conjugates (ADC). The linker (R^(L′))may be stable outside a cell, i.e. extracellular, or it may be cleavableby enzymatic activity, hydrolysis, or other metabolic conditions.Antibody-drug conjugates (ADC) can be conveniently prepared using alinker having reactive functionality for binding to the drug moiety andto the antibody. A cysteine thiol, or an amine, e.g. N-terminus or aminoacid side chain such as lysine, of the antibody (Ab) can form a bondwith a functional group of a linker or spacer reagent, PBD drug moiety(D) or drug-linker reagent (D^(L), D-R^(L)), where R^(L) can be R^(L1)or R^(L2).

The linkers of the ADC preferably prevent aggregation of ADC moleculesand keep the ADC freely soluble in aqueous media and in a monomericstate.

The linkers of the ADC are preferably stable extracellularly. Beforetransport or delivery into a cell, the antibody-drug conjugate (ADC) ispreferably stable and remains intact, i.e. the antibody remains linkedto the drug moiety. The linkers are stable outside the target cell andmay be cleaved at some efficacious rate inside the cell. An effectivelinker will: (i) maintain the specific binding properties of theantibody; (ii) allow intracellular delivery of the conjugate or drugmoiety; (iii) remain stable and intact, i.e. not cleaved, until theconjugate has been delivered or transported to its targeted site; and(iv) maintain a cytotoxic, cell-killing effect or a cytostatic effect ofthe PBD drug moiety. Stability of the ADC may be measured by standardanalytical techniques such as mass spectroscopy, HPLC, and theseparation/analysis technique LC/MS.

Covalent attachment of the antibody and the drug moiety requires thelinker to have two reactive functional groups, i.e. bivalency in areactive sense. Bivalent linker reagents which are useful to attach twoor more functional or biologically active moieties, such as peptides,nucleic acids, drugs, toxins, antibodies, haptens, and reporter groupsare known, and methods have been described their resulting conjugates(Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: NewYork, p 234-242).

In another embodiment, the linker may be substituted with groups whichmodulate aggregation, solubility or reactivity. For example, a sulfonatesubstituent may increase water solubility of the reagent and facilitatethe coupling reaction of the linker reagent with the antibody or thedrug moiety, or facilitate the coupling reaction of Ab-L with D^(L), orD^(L)-L with Ab, depending on the synthetic route employed to preparethe ADC.

In one embodiment, L-R^(L′) is a group:

-   -   where the asterisk indicates the point of attachment to the Drug        Unit (D), Ab is the antibody (L), L¹ is a linker, A is a        connecting group connecting L¹ to the antibody, L² is a covalent        bond or together with —OC(═O)— forms a self-immolative linker,        and L¹ or L² is a cleavable linker.

L¹ is preferably the cleavable linker, and may be referred to as atrigger for activation of the linker for cleavage.

The nature of L¹ and L², where present, can vary widely. These groupsare chosen on the basis of their cleavage characteristics, which may bedictated by the conditions at the site to which the conjugate isdelivered. Those linkers that are cleaved by the action of enzymes arepreferred, although linkers that are cleavable by changes in pH (e.g.acid or base labile), temperature or upon irradiation (e.g. photolabile)may also be used. Linkers that are cleavable under reducing or oxidisingconditions may also find use in the present invention.

L¹ may comprise a contiguous sequence of amino acids. The amino acidsequence may be the target substrate for enzymatic cleavage, therebyallowing release of L-R^(L′) from the N10 position.

In one embodiment, L¹ is cleavable by the action of an enzyme. In oneembodiment, the enzyme is an esterase or a peptidase.

In one embodiment, L² is present and together with —C(═O)O— forms aself-immolative linker. In one embodiment, L² is a substrate forenzymatic activity, thereby allowing release of L-R^(L′) from the N10position.

In one embodiment, where L¹ is cleavable by the action of an enzyme andL² is present, the enzyme cleaves the bond between L¹ and L².

L¹ and L², where present, may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

An amino group of L¹ that connects to L² may be the N-terminus of anamino acid or may be derived from an amino group of an amino acid sidechain, for example a lysine amino acid side chain.

A carboxyl group of L¹ that connects to L² may be the C-terminus of anamino acid or may be derived from a carboxyl group of an amino acid sidechain, for example a glutamic acid amino acid side chain.

A hydroxyl group of L¹ that connects to L² may be derived from ahydroxyl group of an amino acid side chain, for example a serine aminoacid side chain.

The term “amino acid side chain” includes those groups found in: (i)naturally occurring amino acids such as alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine; (ii) minor amino acids suchas ornithine and citrulline; (iii) unnatural amino acids, beta-aminoacids, synthetic analogs and derivatives of naturally occurring aminoacids; and (iv) all enantiomers, diastereomers, isomerically enriched,isotopically labelled (e.g. ²H, ³H, ¹⁴C, ¹⁵N), protected forms, andracemic mixtures thereof.

In one embodiment, —C(═O)O— and L² together form the group:

-   -   where the asterisk indicates the point of attachment to the N10        position, the wavy line indicates the point of attachment to the        linker L¹, Y is —N(H)—, —O—, —C(═O)N(H)— or —C(═O)O—, and n is 0        to 3. The phenylene ring is optionally substituted with one, two        or three substituents as described herein. In one embodiment,        the phenylene group is optionally substituted with halo, NO₂, R        or OR.

In one embodiment, Y is NH.

In one embodiment, n is 0 or 1. Preferably, n is 0.

Where Y is NH and n is 0, the self-immolative linker may be referred toas a p-aminobenzylcarbonyl linker (PABC).

The self-immolative linker will allow for release of the protectedcompound when a remote site is activated, proceeding along the linesshown below (for n=0):

-   -   where L* is the activated form of the remaining portion of the        linker. These groups have the advantage of separating the site        of activation from the compound being protected. As described        above, the phenylene group may be optionally substituted.

In one embodiment described herein, the group L* is a linker L¹ asdescribed herein, which may include a dipeptide group.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above. Each phenylene ring is optionally substituted with one,        two or three substituents as described herein. In one        embodiment, the phenylene ring having the Y substituent is        optionally substituted and the phenylene ring not having the Y        substituent is unsubstituted. In one embodiment, the phenylene        ring having the Y substituent is unsubstituted and the phenylene        ring not having the Y substituent is optionally substituted.

In another embodiment, —C(═O)O— and L² together form a group selectedfrom:

-   -   where the asterisk, the wavy line, Y, and n are as defined        above, E is O, S or NR, D is N, CH, or CR, and F is N, CH, or        CR.

In one embodiment, D is N.

In one embodiment, D is CH.

In one embodiment, E is O or S.

In one embodiment, F is CH.

In a preferred embodiment, the linker is a cathepsin labile linker.

In one embodiment, L¹ comprises a dipeptide The dipeptide may berepresented as —NH—X₁—X₂—CO—, where —NH— and —CO— represent the N- andC-terminals of the amino acid groups X₁ and X₂ respectively. The aminoacids in the dipeptide may be any combination of natural amino acids.Where the linker is a cathepsin labile linker, the dipeptide may be thesite of action for cathepsin-mediated cleavage.

Additionally, for those amino acids groups having carboxyl or amino sidechain functionality, for example Glu and Lys respectively, CO and NH mayrepresent that side chain functionality.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-,    -   -Trp-Cit-        where Cit is citrulline.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X, —X₂—CO—, is-Phe-Lys- or -Val-Ala-.

Other dipeptide combinations may be used, including those described byDubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which isincorporated herein by reference.

In one embodiment, the amino acid side chain is derivatised, whereappropriate. For example, an amino group or carboxy group of an aminoacid side chain may be derivatised. In one embodiment, an amino groupNH₂ of a side chain amino acid, such as lysine, is a derivatised formselected from the group consisting of NHR and NRR′.

In one embodiment, a carboxy group COOH of a side chain amino acid, suchas aspartic acid, is a derivatised form selected from the groupconsisting of COOR, CONH₂, CONHR and CONRR′.

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed below in relation to the group R^(L). The present inventorshave established that protected amino acid sequences are cleavable byenzymes. For example, it has been established that a dipeptide sequencecomprising a Boc side chain-protected Lys residue is cleavable bycathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog. Additionalprotecting group strategies are set out in Protective Groups in OrganicSynthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those aminoacids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;    -   Asn: Trt, Xan;    -   Asp: Bzl, t-Bu;    -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;    -   Glu: Bzl, t-Bu;    -   Gin: Trt, Xan;    -   His: Boc, Dnp, Tos, Trt;    -   Lys: Boc, Z—Cl, Fmoc, Z, Alloc;    -   Ser: Bzl, TBDMS, TBDPS;    -   Thr: Bz;    -   Trp: Boc;    -   Tyr: Bzl, Z, Z—Br.

In one embodiment, the side chain protection is selected to beorthogonal to a group provided as, or as part of, a capping group, wherepresent. Thus, the removal of the side chain protecting group does notremove the capping group, or any protecting group functionality that ispart of the capping group.

In other embodiments of the invention, the amino acids selected arethose having no reactive side chain functionality. For example, theamino acids may be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro, andVal.

In one embodiment, the dipeptide is used in combination with aself-immolative linker. The self-immolative linker may be connected to—X₂—.

Where a self-immolative linker is present, —X₂— is connected directly tothe self-immolative linker. Preferably the group —X₂—CO— is connected toY, where Y is NH, thereby forming the group —X₂—CO—NH—.

—NH—X₁— is connected directly to A. A may comprise the functionality—CO— thereby to form an amide link with —X₁—.

In one embodiment, L¹ and L² together with —OC(═O)— comprise the groupNH—X₁—X₂—CO-PABC-. The PABC group is connected directly to the N10position.

Preferably, the self-immolative linker and the dipeptide together formthe group —NH-Phe-Lys-CO—NH-PABC-, which is illustrated below:

-   -   where the asterisk indicates the point of attachment to the N10        position, and the wavy line indicates the point of attachment to        the remaining portion of the linker L¹ or the point of        attachment to A. Preferably, the wavy line indicates the point        of attachment to A. The side chain of the Lys amino acid may be        protected, for example, with Boc, Fmoc, or Alloc, as described        above.

Alternatively, the self-immolative linker and the dipeptide togetherform the group —NH-Val-Ala-CO—NH-PABC-, which is illustrated below:

-   -   where the asterisk and the wavy line are as defined above.

Alternatively, the self-immolative linker and the dipeptide togetherform the group —NH-Val-Cit-CO—NH-PABC-, which is illustrated below:

-   -   where the asterisk and the wavy line are as defined above.

In one embodiment, A is a covalent bond. Thus, L¹ and the antibody aredirectly connected. For example, where L¹ comprises a contiguous aminoacid sequence, the N-terminus of the sequence may connect directly tothe antibody.

Thus, where A is a covalent bond, the connection between the antibodyand L¹ may be selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —C(═O)NHC(═O)—,    -   —S—,    -   —S—S—,    -   —CH₂C(═O)—, and    -   ═N—NH—.

An amino group of L¹ that connects to the antibody may be the N-terminusof an amino acid or may be derived from an amino group of an amino acidside chain, for example a lysine amino acid side chain.

An carboxyl group of L¹ that connects to the antibody may be theC-terminus of an amino acid or may be derived from a carboxyl group ofan amino acid side chain, for example a glutamic acid amino acid sidechain.

A hydroxyl group of L¹ that connects to the antibody may be derived froma hydroxyl group of an amino acid side chain, for example a serine aminoacid side chain.

A thiol group of L¹ that connects to the antibody may be derived from athiol group of an amino acid side chain, for example a serine amino acidside chain.

The comments above in relation to the amino, carboxyl, hydroxyl andthiol groups of L¹ also apply to the antibody.

In one embodiment, L² together with —OC(═O)— represents:

-   -   where the asterisk indicates the point of attachment to the N10        position, the wavy line indicates the point of attachment to L¹,        n is 0 to 3, Y is a covalent bond or a functional group, and E        is an activatable group, for example by enzymatic action or        light, thereby to generate a self-immolative unit. The phenylene        ring is optionally further substituted with one, two or three        substituents as described herein. In one embodiment, the        phenylene group is optionally further substituted with halo,        NO₂, R or OR. Preferably n is 0 or 1, most preferably 0.

E is selected such that the group is susceptible to activation, e.g. bylight or by the action of an enzyme. E may be —NO₂ or glucoronic acid.The former may be susceptible to the action of a nitroreductase, thelatter to the action of a β-glucoronidase.

In this embodiment, the self-immolative linker will allow for release ofthe protected compound when E is activated, proceeding along the linesshown below (for n=0):

-   -   where the asterisk indicates the point of attachment to the N10        position, E is the activated form of E, and Y is as described        above. These groups have the advantage of separating the site of        activation from the compound being protected. As described        above, the phenylene group may be optionally further        substituted.

The group Y may be a covalent bond to L¹.

The group Y may be a functional group selected from:

-   -   —C(═O)—    -   —NH—    -   —O—    -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—, and    -   —S—.

Where L¹ is a dipeptide, it is preferred that Y is —NH— or —C(═O)—,thereby to form an amide bond between L¹ and Y. In this embodiment, thedipeptide sequence need not be a substrate for an enzymatic activity.

In another embodiment, A is a spacer group. Thus, L¹ and the antibodyare indirectly connected.

L¹ and A may be connected by a bond selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—, and    -   —NHC(═O)NH—.

In one embodiment, the group A is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the antibody, and        n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the antibody, and        n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the group A is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the antibody, n        is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably        4 or 8. In another embodiment, m is 10 to 30, and preferably 20        to 30. Alternatively, m is 0 to 50. In this embodiment, m is        preferably 10-40 and n is 1.

In one embodiment, the group A is:

-   -   where the asterisk indicates the point of attachment to L¹, the        wavy line indicates the point of attachment to the antibody, n        is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, and most preferably        4 or 8. In another embodiment, m is 10 to 30, and preferably 20        to 30. Alternatively, m is 0 to 50. In this embodiment, m is        preferably 10-40 and n is 1.

In one embodiment, the connection between the antibody and A is througha thiol residue of the antibody and a maleimide group of A.

In one embodiment, the connection between the antibody and A is:

-   -   where the asterisk indicates the point of attachment to the        remaining portion of A and the wavy line indicates the point of        attachment to the remaining portion of the antibody. In this        embodiment, the S atom is typically derived from the antibody.

In each of the embodiments above, an alternative functionality may beused in place of the maleimide-derived group shown below:

-   -   where the wavy line indicates the point of attachment to the        antibody as before, and the asterisk indicates the bond to the        remaining portion of the A group.

In one embodiment, the maleimide-derived group is replaced with thegroup:

-   -   where the wavy line indicates point of attachment to the        antibody, and the asterisk indicates the bond to the remaining        portion of the A group.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the antibody, is selected from:

-   -   —C(═O)NH—,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)NH—,    -   —NHC(═O)NH—,    -   —NHC(═O)NH,    -   —C(═O)NHC(═O)—,    -   —S—,    -   —S—S—,    -   —CH₂C(═O)—    -   —C(═O)CH₂—,    -   ═N—NH—, and    -   —NH—N═.

In one embodiment, the maleimide-derived group is replaced with a group,which optionally together with the antibody, is selected from:

-   -   where the wavy line indicates either the point of attachment to        the antibody or the bond to the remaining portion of the A        group, and the asterisk indicates the other of the point of        attachment to the antibody or the bond to the remaining portion        of the A group.

Other groups suitable for connecting L¹ to the antibody are described inWO 2005/082023.

In one embodiment, the Connecting Group A is present, the Trigger L¹ ispresent and Self-Immolative Linker L² is absent. Thus, L¹ and the Drugunit are directly connected via a bond. Equivalently in this embodiment,L² is a bond. This may be particularly relevant when D^(L) is of FormulaII.

L¹ and D may be connected by a bond selected from:

-   -   —C(═O)N<,    -   —C(═O)O—,    -   —NHC(═O)—,    -   —OC(═O)—,    -   —OC(═O)O—,    -   —NHC(═O)O—,    -   —OC(═O)N<, and    -   —NHC(═O)N<,        where N< or O— are part of D.

In one embodiment, L¹ and D are preferably connected by a bond selectedfrom:

-   -   —C(═O)N<, and    -   —NHC(═O)—.

In one embodiment, L¹ comprises a dipeptide and one end of the dipeptideis linked to D. As described above, the amino acids in the dipeptide maybe any combination of natural amino acids and non-natural amino acids.In some embodiments, the dipeptide comprises natural amino acids. Wherethe linker is a cathepsin labile linker, the dipeptide is the site ofaction for cathepsin-mediated cleavage. The dipeptide then is arecognition site for cathepsin.

In one embodiment, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, isselected from:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-,    -   -Val-Cit-,    -   -Phe-Cit-,    -   -Leu-Cit-,    -   -Ile-Cit-,    -   -Phe-Arg-, and    -   -Trp-Cit-;        where Cit is citrulline. In such a dipeptide, —NH— is the amino        group of X₁, and CO is the carbonyl group of X₂.

Preferably, the group —X₁—X₂— in dipeptide, —NH—X₁—X₂—CO—, is selectedfrom:

-   -   -Phe-Lys-,    -   -Val-Ala-,    -   -Val-Lys-,    -   -Ala-Lys-, and    -   -Val-Cit-.

Most preferably, the group —X₁—X₂— in dipeptide, —NH—X, —X₂—CO—, is-Phe-Lys- or -Val-Ala-.

Other dipeptide combinations of interest include:

-   -   -Gly-Gly-,    -   -Pro-Pro-, and    -   -Val-Glu-.

Other dipeptide combinations may be used, including those describedabove.

In one embodiment, L¹-D is:

-   -   where —NH—X₁—X₂—CO is the dipeptide, —N< is part of the Drug        unit, the asterisk indicates the points of attachment to the        remainder of the Drug unit, and the wavy line indicates the        point of attachment to the remaining portion of L¹ or the point        of attachment to A. Preferably, the wavy line indicates the        point of attachment to A.

In one embodiment, the dipeptide is valine-alanine and L¹-D is:

-   -   where the asterisks, —N< and the wavy line are as defined above.

In one embodiment, the dipeptide is phenylalnine-lysine and L¹-D is:

-   -   where the asterisks, —N< and the wavy line are as defined above.

In one embodiment, the dipeptide is valine-citrulline.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 7, preferably 3 to 7, most        preferably 3 or 7.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A-L¹ is:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 8, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the rest of the Ligand unit, and n is        0 to 6. In one embodiment, n is 5.

In one embodiment, the group A-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the remainder of the Ligand unit, and        n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        S is a sulfur group of the Ligand unit, the wavy line indicates        the point of attachment to the remainder of the Ligand unit, n        is 0 or 1, and m is 0 to 30. In a preferred embodiment, n is 1        and m is 0 to 10, 1 to 8, preferably 4 to 8, most preferably 4        or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the Ligand        unit, n is 0 or 1, and m is 0 to 30. In a preferred embodiment,        n is 1 and m is 0 to 10, 1 to 7, preferably 4 to 8, most        preferably 4 or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, and n is 0 to 6. In one embodiment, n is 5.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

In one embodiment, the groups A¹-L¹ are:

-   -   where the asterisk indicates the point of attachment to L² or D,        the wavy line indicates the point of attachment to the remainder        of the Ligand unit, n is 0 or 1, and m is 0 to 30. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 8,        preferably 4 to 8, most preferably 4 or 8.

The group R^(L′) is derivable from the group R^(L). The group R^(L) maybe converted to a group R^(L′) by connection of an antibody to afunctional group of R^(L). Other steps may be taken to convert R^(L) toR^(L′). These steps may include the removal of protecting groups, wherepresent, or the installation of an appropriate functional group.

R^(L)

Linkers can include protease-cleavable peptidic moieties comprising oneor more amino acid units. Peptide linker reagents may be prepared bysolid phase or liquid phase synthesis methods (E. Schröder and K. Lübke,The Peptides, volume 1, pp 76-136 (1965) Academic Press) that are wellknown in the field of peptide chemistry, including t-BOC chemistry(Geiser et al “Automation of solid-phase peptide synthesis” inMacromolecular Sequencing and Synthesis, Alan R. Liss, Inc., 1988, pp.199-218) and Fmoc/HBTU chemistry (Fields, G. and Noble, R. (1990) “Solidphase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl aminoacids”, Int. J. Peptide Protein Res. 35:161-214), on an automatedsynthesizer such as the Rainin Symphony Peptide Synthesizer (ProteinTechnologies, Inc., Tucson, Ariz.), or Model 433 (Applied Biosystems,Foster City, Calif.).

Exemplary amino acid linkers include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Amino acid side chains include those occurring naturally, as well asminor amino acids and non-naturally occurring amino acid analogs, suchas citrulline. Amino acid side chains include hydrogen, methyl,isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, as well as thefollowing structures:

When the amino acid side chains include other than hydrogen (glycine),the carbon atom to which the amino acid side chain is attached ischiral. Each carbon atom to which the amino acid side chain is attachedis independently in the (S) or (R) configuration, or a racemic mixture.Drug-linker reagents may thus be enantiomerically pure, racemic, ordiastereomeric.

In exemplary embodiments, amino acid side chains are selected from thoseof natural and non-natural amino acids, including alanine,2-amino-2-cyclohexylacetic acid, 2-amino-2-phenylacetic acid, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, norleucine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,γ-aminobutyric acid, α,α-dimethyl γ-aminobutyric acid, β,β-dimethylγ-aminobutyric acid, ornithine, and citrulline (Cit).

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagentuseful for constructing a linker-PBD drug moiety intermediate forconjugation to an antibody, having a para-aminobenzylcarbamoyl (PAB)self-immolative spacer has the structure:

where Q is C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, —NO₂ or —CN; and mis an integer ranging from 0-4.

An exemplary phe-lys(Mtr) dipeptide linker reagent having ap-aminobenzyl group can be prepared according to Dubowchik, et al.(1997) Tetrahedron Letters, 38:5257-60, and has the structure:

where Mtr is mono-4-methoxytrityl, Q is C₁-C₈ alkyl, —O—(C₁-C₈ alkyl),-halogen, —NO₂ or —CN; and m is an integer ranging from 0-4.

The “self-immolative linker” PAB (para-aminobenzyloxycarbonyl), attachesthe drug moiety to the antibody in the antibody drug conjugate (Carl etal (1981) J. Med. Chem. 24:479-480; Chakravarty et al (1983) J. Med.Chem. 26:638-644; U.S. Pat. No. 6,214,345; US20030130189; US20030096743;U.S. Pat. No. 6,759,509; US20040052793; U.S. Pat. No. 6,218,519; U.S.Pat. No. 6,835,807; U.S. Pat. No. 6,268,488; US20040018194; WO98/13059;US20040052793; U.S. Pat. No. 6,677,435; U.S. Pat. No. 5,621,002;US20040121940; WO2004/032828). Other examples of self-immolative spacersbesides PAB include, but are not limited to: (i) aromatic compounds thatare electronically similar to the PAB group such as2-aminoimidazol-5-methanol derivatives (Hay et al. (1999) Bioorg. Med.Chem. Lett. 9:2237), thiazoles (U.S. Pat. No. 7,375,078), multiple,elongated PAB units (de Groot et al (2001) J. Org. Chem. 66:8815-8830);and ortho or para-aminobenzylacetals; and (ii) homologated styryl PABanalogs (U.S. Pat. No. 7,223,837). Spacers can be used that undergocyclization upon amide bond hydrolysis, such as substituted andunsubstituted 4-aminobutyric acid amides (Rodrigues et al (1995)Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] andbicyclo[2.2.2] ring systems (Storm et al (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al (1990)J. Org. Chem. 55:5867). Elimination of amine-containing drugs that aresubstituted at glycine (Kingsbury et al (1984) J. Med. Chem. 27:1447)are also examples of self-immolative spacers useful in ADC.

In one embodiment, a valine-citrulline dipeptide PAB analog reagent hasa 2,6 dimethyl phenyl group and has the structure:

Linker reagents useful for the antibody drug conjugates of the inventioninclude, but are not limited to: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and bis-maleimide reagents:DTME, BMB, BMDB, BMH, BMOE, 1,8-bis-maleimidodiethyleneglycol(BM(PEO)₂), and 1,11-bis-maleimidotriethyleneglycol (BM(PEO)₃), whichare commercially available from Pierce Biotechnology, Inc.,ThermoScientific, Rockford, Ill., and other reagent suppliers.Bis-maleimide reagents allow the attachment of a free thiol group of acysteine residue of an antibody to a thiol-containing drug moiety,label, or linker intermediate, in a sequential or concurrent fashion.Other functional groups besides maleimide, which are reactive with athiol group of an antibody, PBD drug moiety, or linker intermediateinclude iodoacetamide, bromoacetamide, vinyl pyridine, disulfide,pyridyl disulfide, isocyanate, and isothiocyanate.

Other embodiments of linker reagents are:N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP),N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP, Carlsson et al(1978) Biochem. J. 173:723-737),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Useful linker reagents can also beobtained via other commercial sources, such as Molecular BiosciencesInc. (Boulder, Colo.), or synthesized in accordance with proceduresdescribed in Toki et al (2002) J. Org. Chem. 67:1866-1872; U.S. Pat. No.6,214,345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO03/043583; and WO 04/032828.

The Linker may be a dendritic type linker for covalent attachment ofmore than one drug moiety through a branching, multifunctional linkermoiety to an antibody (US 2006/116422; US 2005/271615; de Groot et al(2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al (2003) Angew.Chem. Int. Ed. 42:4494-4499; Shamis et al (2004) J. Am. Chem. Soc.126:1726-1731; Sun et al (2002) Bioorganic & Medicinal Chemistry Letters12:2213-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry11:1761-1768; King et al (2002) Tetrahedron Letters 43:1987-1990).Dendritic linkers can increase the molar ratio of drug to antibody, i.e.loading, which is related to the potency of the ADC. Thus, where anantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic or branched linker.

One exemplary embodiment of a dendritic type linker has the structure:

where the asterisk indicate the point of attachment to the N10 positionof a PBD moiety.R^(C), Capping Group

The conjugate of the first aspect of the invention may have a cappinggroup R^(C) at the N10 position. Compound E may have a capping groupR^(C).

In one embodiment, where the conjugate is a dimer with each monomerbeing of formula (A), the group R¹⁰ in one of the monomer units is acapping group R^(C) or is a group R¹⁰.

In one embodiment, where the conjugate is a dimer with each monomerbeing of formula (A), the group R¹⁰ in one of the monomer units is acapping group R^(C).

In one embodiment, where compound E is a dimer with each monomer beingof formula (E), the group R^(L) in one of the monomer units is a cappinggroup R^(C) or is a linker for connection to an antibody.

In one embodiment, where compound E is a dimer with each monomer beingof formula (E), the group R^(L) in one of the monomer units is a cappinggroup R^(C).

The group R^(C) is removable from the N10 position of the PBD moiety toleave an N10-C11 imine bond, a carbinolamine, a substitutedcarbinolamine, where QR¹¹ is OSO₃M, a bisulfite adduct, athiocarbinolamine, a substituted thiocarbinolamine, or a substitutedcarbinalamine.

In one embodiment, R^(C), may be a protecting group that is removable toleave an N10-C11 imine bond, a carbinolamine, a substitutedcabinolamine, or, where QR¹¹ is OSO₃M, a bisulfite adduct. In oneembodiment, R^(C) is a protecting group that is removable to leave anN10-C11 imine bond.

The group R^(c) is intended to be removable under the same conditions asthose required for the removal of the group R¹⁰, for example to yield anN10-C11 imine bond, a carbinolamine and so on. The capping group acts asa protecting group for the intended functionality at the N10 position.The capping group is intended not to be reactive towards an antibody.For example, R^(C) is not the same as R^(L).

Compounds having a capping group may be used as intermediates in thesynthesis of dimers having an imine monomer. Alternatively, compoundshaving a capping group may be used as conjugates, where the cappinggroup is removed at the target location to yield an imine, acarbinolamine, a substituted cabinolamine and so on. Thus, in thisembodiment, the capping group may be referred to as a therapeuticallyremovable nitrogen protecting group, as defined in the inventors'earlier application WO 00/12507.

In one embodiment, the group R^(C) is removable under the conditionsthat cleave the linker R^(L) of the group R¹⁰. Thus, in one embodiment,the capping group is cleavable by the action of an enzyme.

In an alternative embodiment, the capping group is removable prior tothe connection of the linker R^(L) to the antibody. In this embodiment,the capping group is removable under conditions that do not cleave thelinker R^(L).

Where a compound includes a functional group G¹ to form a connection tothe antibody, the capping group is removable prior to the addition orunmasking of G¹.

The capping group may be used as part of a protecting group strategy toensure that only one of the monomer units in a dimer is connected to anantibody.

The capping group may be used as a mask for a N10-C11 imine bond. Thecapping group may be removed at such time as the imine functionality isrequired in the compound. The capping group is also a mask for acarbinolamine, a substituted cabinolamine, and a bisulfite adduct, asdescribed above.

R^(C) may be an N10 protecting group, such as those groups described inthe inventors' earlier application, WO 00/12507. In one embodiment,R^(C) is a therapeutically removable nitrogen protecting group, asdefined in the inventors' earlier application, WO 00/12507.

In one embodiment, R^(C) is a carbamate protecting group.

In one embodiment, the carbamate protecting group is selected from:

-   -   Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.

Optionally, the carbamate protecting group is further selected from Moc.

In one embodiment, R^(C) is a linker group R^(L) lacking the functionalgroup for connection to the antibody.

This application is particularly concerned with those R^(C) groups whichare carbamates.

In one embodiment, R^(C) is a group:

-   -   where the asterisk indicates the point of attachment to the N10        position, G² is a terminating group, L³ is a covalent bond or a        cleavable linker L¹, L² is a covalent bond or together with        OC(═O) forms a self-immolative linker.

Where L³ and L² are both covalent bonds, G² and OC(═O) together form acarbamate protecting group as defined above.

L¹ is as defined above in relation to R¹⁰.

L² is as defined above in relation to R¹⁰.

Various terminating groups are described below, including those based onwell known protecting groups.

In one embodiment L³ is a cleavable linker L¹, and L², together withOC(═O), forms a self-immolative linker. In this embodiment, G² is Ac(acetyl) or Moc, or a carbamate protecting group selected from:

-   -   Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.

Optionally, the carbamate protecting group is further selected from Moc.

In another embodiment, G² is an acyl group —C(═O)G³, where G³ isselected from alkyl (including cycloalkyl, alkenyl and alkynyl),heteroalkyl, heterocyclyl and aryl (including heteroaryl and carboaryl).These groups may be optionally substituted. The acyl group together withan amino group of L³ or L², where appropriate, may form an amide bond.The acyl group together with a hydroxy group of L³ or L², whereappropriate, may form an ester bond.

In one embodiment, G³ is heteroalkyl. The heteroalkyl group may comprisepolyethylene glycol. The heteroalkyl group may have a heteroatom, suchas O or N, adjacent to the acyl group, thereby forming a carbamate orcarbonate group, where appropriate, with a heteroatom present in thegroup L³ or L², where appropriate.

In one embodiment, G³ is selected from NH₂, NHR and NRR′. Preferably, G³is NRR′.

In one embodiment G² is the group:

-   -   where the asterisk indicates the point of attachment to L³, n is        0 to 6 and G⁴ is selected from OH, OR, SH, SR, COOR, CONH₂,        CONHR, CONRR′, NH₂, NHR, NRR′, NO₂, and halo. The groups OH, SH,        NH₂ and NHR are protected. In one embodiment, n is 1 to 6, and        preferably n is 5. In one embodiment, G⁴ is OR, SR, COOR, CONH₂,        CONHR, CONRR′, and NRR′. In one embodiment, G⁴ is OR, SR, and        NRR′. Preferably G⁴ is selected from OR and NRR′, most        preferably G⁴ is OR. Most preferably G⁴ is OMe.

In one embodiment, the group G² is:

-   -   where the asterisk indicates the point of attachment to L³, and        n and G⁴ are as defined above.

In one embodiment, the group G² is:

-   -   where the asterisk indicates the point of attachment to L³, n is        0 or 1, m is 0 to 50, and G⁴ is selected from OH, OR, SH, SR,        COOR, CONH₂, CONHR, CONRR′, NH₂, NHR, NRR′, NO₂, and halo. In a        preferred embodiment, n is 1 and m is 0 to 10, 1 to 2,        preferably 4 to 8, and most preferably 4 or 8. In another        embodiment, n is 1 and m is 10 to 50, preferably 20 to 40. The        groups OH, SH, NH₂ and NHR are protected. In one embodiment, G⁴        is OR, SR, COOR, CONH₂, CONHR, CONRR′, and NRR′. In one        embodiment, G⁴ is OR, SR, and NRR′. Preferably G⁴ is selected        from OR and NRR′, most preferably G⁴ is OR. Preferably G⁴ is        OMe.

In one embodiment, the group G² is:

-   -   where the asterisk indicates the point of attachment to L³, and        n, m and G⁴ are as defined above.

In one embodiment, the group G² is:

-   -   where n is 1-20, m is 0-6, and G⁴ is selected from OH, OR, SH,        SR, COOR, CONH₂, CONHR, CONRR′, NH₂, NHR, NRR′, NO₂, and halo.        In one embodiment, n is 1-10. In another embodiment, n is 10 to        50, preferably 20 to 40. In one embodiment, n is 1. In one        embodiment, m is 1. The groups OH, SH, NH₂ and NHR are        protected. In one embodiment, G⁴ is OR, SR, COOR, CONH₂, CONHR,        CONRR′, and NRR′. In one embodiment, G⁴ is OR, SR, and NRR′.        Preferably G⁴ is selected from OR and NRR′, most preferably G⁴        is OR. Preferably G⁴ is OMe.

In one embodiment, the group G² is:

-   -   where the asterisk indicates the point of attachment to L³, and        n, m and G⁴ are as defined above.

In each of the embodiments above G⁴ may be OH, SH, NH₂ and NHR. Thesegroups are preferably protected.

In one embodiment, OH is protected with Bzl, TBDMS, or TBDPS.

In one embodiment, SH is protected with Acm, Bzl, Bzl-OMe, Bzl-Me, orTrt.

In one embodiment, NH₂ or NHR are protected with Boc, Moc, Z—Cl, Fmoc,Z, or Alloc.

In one embodiment, the group G² is present in combination with a groupL³, which group is a dipeptide.

The capping group is not intended for connection to the antibody. Thus,the other monomer present in the dimer serves as the point of connectionto the antibody via a linker. Accordingly, it is preferred that thefunctionality present in the capping group is not available for reactionwith an antibody. Thus, reactive functional groups such as OH, SH, NH₂,COOH are preferably avoided. However, such functionality may be presentin the capping group if protected, as described above.

EMBODIMENTS

Embodiments of the present invention include ConjA wherein the antibodyis as defined above.

Embodiments of the present invention include ConjB wherein the antibodyis as defined above.

Embodiments of the present invention include ConjC wherein the antibodyis as defined above.

Embodiments of the present invention include ConjD wherein the antibodyis as defined above.

Embodiments of the present invention include ConjE wherein the antibodyis as defined above.

As mentioned above, some embodiments of the invention exclude ConjA,ConjB, ConjC, ConjD and ConjE.

Drug Loading

The drug loading is the average number of PBD drugs per antibody, e.g.antibody. Where the compounds of the invention are bound to cysteines,drug loading may range from 1 to 8 drugs (D^(L)) per antibody, i.e.where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attachedto the antibody. Compositions of conjugates include collections ofantibodies, conjugated with a range of drugs, from 1 to 8. Where thecompounds of the invention are bound to lysines, drug loading may rangefrom 1 to 80 drugs (D^(L)) per antibody, although an upper limit of 40,20, 10 or 8 may be preferred. Compositions of conjugates includecollections of antibodies, conjugated with a range of drugs, from 1 to80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, andelectrophoresis. The quantitative distribution of ADC in terms of p mayalso be determined. By ELISA, the averaged value of p in a particularpreparation of ADC may be determined (Hamblett et al (2004) Clin. CancerRes. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852).However, the distribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis. Suchtechniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading, e.g. p >5, may cause aggregation,insolubility, toxicity, or loss of cellular permeability of certainantibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with thedrug-linker intermediate (D-L) or linker reagent. Only the most reactivelysine groups may react with an amine-reactive linker reagent. Also,only the most reactive cysteine thiol groups may react with athiol-reactive linker reagent. Generally, antibodies do not containmany, if any, free and reactive cysteine thiol groups which may belinked to a drug moiety. Most cysteine thiol residues in the antibodiesof the compounds exist as disulfide bridges and must be reduced with areducing agent such as dithiothreitol (DTT) or TCEP, under partial ortotal reducing conditions. The loading (drug/antibody ratio) of an ADCmay be controlled in several different manners, including: (i) limitingthe molar excess of drug-linker intermediate (D-L) or linker reagentrelative to antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification.

Certain antibodies have reducible interchain disulfides, i.e. cysteinebridges. Antibodies may be made reactive for conjugation with linkerreagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by engineering one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541teaches engineering antibodies by introduction of reactive cysteineamino acids.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al(2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No.7,723,485; WO2009/052249). The engineered cysteine thiols may react withlinker reagents or the drug-linker reagents of the present inventionwhich have thiol-reactive, electrophilic groups such as maleimide oralpha-halo amides to form ADC with cysteine engineered antibodies andthe PBD drug moieties. The location of the drug moiety can thus bedesigned, controlled, and known. The drug loading can be controlledsince the engineered cysteine thiol groups typically react withthiol-reactive linker reagents or drug-linker reagents in high yield.Engineering an IgG antibody to introduce a cysteine amino acid bysubstitution at a single site on the heavy or light chain gives two newcysteines on the symmetrical antibody. A drug loading near 2 can beachieved with near homogeneity of the conjugation product ADC.

Alternatively, site-specific conjugation can be achieved by engineeringantibodies to contain unnatural amino acids in their heavy and/or lightchains as described by Axup et al. ((2012), Proc Natl Acad Sci USA.109(40):16101-16116). The unnatural amino acids provide the additionaladvantage that orthogonal chemistry can be designed to attach the linkerreagent and drug.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

Thus the antibody-drug conjugate compositions of the invention includemixtures of antibody-drug conjugate compounds where the antibody has oneor more PBD drug moieties and where the drug moieties may be attached tothe antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepinegroups per antibody is in the range 1 to 20. In some embodiments therange is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.

In some embodiments, there is one dimer pyrrolobenzodiazepine group perantibody.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N+HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K+, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R²⁺, NHR₃⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are thosederived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acidand valeric. Examples of suitable polymeric organic anions include, butare not limited to, those derived from the following polymeric acids:tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

The invention includes compounds where a solvent adds across the iminebond of the PBD moiety, which is illustrated below where the solvent iswater or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD (as described in the section relating to R¹⁰ above).The balance of these equilibria depend on the conditions in which thecompounds are found, as well as the nature of the moiety itself.

These particular compounds may be isolated in solid form, for example,by lyophilisation.

Isomers

Certain compounds of the invention may exist in one or more particulargeometric, optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or l meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to ²H(deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶C, and ¹²⁵I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labelled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labelled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent. The concentration of such aheavier isotope, specifically deuterium, may be defined by an isotopicenrichment factor. In the compounds of this invention any atom notspecifically designated as a particular isotope is meant to representany stable isotope of that atom.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Biological Activity

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells having receptorproteins to the antibody of the ADC in a cell culture medium; culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability. Cell-based in vitro assays are used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of an ADC of the invention.

The in vitro potency of antibody-drug conjugates can be measured by acell proliferation assay. The CellTiter-Glo® Luminescent Cell ViabilityAssay is a commercially available (Promega Corp., Madison, Wis.),homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and5,700,670). This cell proliferation assay determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al (1993) J. Immunol.Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay isconducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing. The cells may be treated continuously with ADC, or they may betreated and separated from ADC. Generally, cells treated briefly, i.e. 3hours, showed the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons.

The in vitro potency of antibody-drug conjugates can also be measured bya cytotoxicity assay. Cultured adherent cells are washed with PBS,detached with trypsin, diluted in complete medium, containing 10% FCS,centrifuged, re-suspended in fresh medium and counted with ahaemocytometer. Suspension cultures are counted directly. Monodispersecell suspensions suitable for counting may require agitation of thesuspension by repeated aspiration to break up cell clumps.

The cell suspension is diluted to the desired seeding density anddispensed (100 μl per well) into black 96 well plates. Plates ofadherent cell lines are incubated overnight to allow adherence.Suspension cell cultures can be used on the day of seeding.

A stock solution (1 ml) of ADC (20 μg/ml) is made in the appropriatecell culture medium. Serial 10-fold dilutions of stock ADC are made in15 ml centrifuge tubes by serially transferring 100 μl to 900 μl of cellculture medium.

Four replicate wells of each ADC dilution (100 μl) are dispensed in96-well black plates, previously plated with cell suspension (100 μl),resulting in a final volume of 200 μl. Control wells receive cellculture medium (100 μl).

If the doubling time of the cell line is greater than 30 hours, ADCincubation is for 5 days, otherwise a four day incubation is done.

At the end of the incubation period, cell viability is assessed with theAlamar blue assay. AlamarBlue (Invitrogen) is dispensed over the wholeplate (20 μl per well) and incubated for 4 hours. Alamar bluefluorescence is measured at excitation 570 nm, emission 585 nm on theVarioskan flash plate reader. Percentage cell survival is calculatedfrom the mean fluorescence in the ADC treated wells compared to the meanfluorescence in the control wells.

Use

The conjugates of the invention may be used to provide a PBD compound ata target location.

The target location is preferably a proliferative cell population. Theantibody is an antibody for an antigen present on a proliferative cellpopulation.

In one embodiment the antigen is absent or present at a reduced level ina non-proliferative cell population compared to the amount of antigenpresent in the proliferative cell population, for example a tumour cellpopulation.

At the target location the linker may be cleaved so as to release acompound RelA, RelB, RelC, RelD or RelE. Thus, the conjugate may be usedto selectively provide a compound RelA, RelB, Rel C, RelD or RelE to thetarget location.

The linker may be cleaved by an enzyme present at the target location.

The target location may be in vitro, in vivo or ex vivo.

The antibody-drug conjugate (ADC) compounds of the invention includethose with utility for anticancer activity. In particular, the compoundsinclude an antibody conjugated, i.e. covalently attached by a linker, toa PBD drug moiety, i.e. toxin. When the drug is not conjugated to anantibody, the PBD drug has a cytotoxic effect. The biological activityof the PBD drug moiety is thus modulated by conjugation to an antibody.The antibody-drug conjugates (ADC) of the invention selectively deliveran effective dose of a cytotoxic agent to tumor tissue whereby greaterselectivity, i.e. a lower efficacious dose, may be achieved.

Thus, in one aspect, the present invention provides a conjugate compoundas described herein for use in therapy.

In a further aspect there is also provides a conjugate compound asdescribed herein for use in the treatment of a proliferative disease. Asecond aspect of the present invention provides the use of a conjugatecompound in the manufacture of a medicament for treating a proliferativedisease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g. histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias,psoriasis, bone diseases, fibroproliferative disorders (e.g. ofconnective tissues), and atherosclerosis. Cancers of particular interestinclude, but are not limited to, leukemias and ovarian cancers.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

Disorders of particular interest include, but are not limited to,non-Hodgkin Lymphoma including diffuse large B-cell lymphoma (DLBCL),follicular lymphoma, (FL), Mantle Cell lymphoma (MCL), chronic lymphaticlymphoma (CLL) nd leukemias such as Hairy cell leukemia (HCL), Hairycell leukemia variant (HCL-v) and Acute Lymphoblastic Leukaemia (ALL).

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors; leukemia, haematological, and lymphoid malignancies. Othersinclude neuronal, glial, astrocytal, hypothalamic, glandular,macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenicand immunologic, including autoimmune, disorders.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatmentinclude rheumatologic disorders (such as, for example, rheumatoidarthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupusnephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),osteoarthritis, autoimmune gastrointestinal and liver disorders (suchas, for example, inflammatory bowel diseases (e.g. ulcerative colitisand Crohn's disease), autoimmune gastritis and pernicious anemia,autoimmune hepatitis, primary biliary cirrhosis, primary sclerosingcholangitis, and celiac disease), vasculitis (such as, for example,ANCA-associated vasculitis, including Churg-Strauss vasculitis,Wegener's granulomatosis, and polyarteriitis), autoimmune neurologicaldisorders (such as, for example, multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), renaldisorders (such as, for example, glomerulonephritis, Goodpasture'ssyndrome, and Berger's disease), autoimmune dermatologic disorders (suchas, for example, psoriasis, urticaria, hives, pemphigus vulgaris,bullous pemphigoid, and cutaneous lupus erythematosus), hematologicdisorders (such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g. Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

Methods of Treatment

The conjugates of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a conjugate compound of theinvention. The term “therapeutically effective amount” is an amountsufficient to show benefit to a patient. Such benefit may be at leastamelioration of at least one symptom. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of what is being treated. Prescription of treatment, e.g.decisions on dosage, is within the responsibility of generalpractitioners and other medical doctors.

A compound of the invention may be administered alone or in combinationwith other treatments, either simultaneously or sequentially dependentupon the condition to be treated. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g. drugs, such as chemotherapeutics);surgery; and radiation therapy.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, calicheamicin gamma1I, calicheamicin omegal1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),ofatumumab (ARZERRA®, GSK), pertuzumab (PERJETA™, OMNITARG™, 2C4,Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar,Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin(MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the conjugates of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a conjugate compound, a pharmaceuticallyacceptable excipient, carrier, buffer, stabiliser or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material willdepend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Formulations

While it is possible for the conjugate compound to be used (e.g.,administered) alone, it is often preferable to present it as acomposition or formulation.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a conjugatecompound, as described herein, and a pharmaceutically acceptablecarrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one conjugate compound, as described herein,together with one or more other pharmaceutically acceptable ingredientswell known to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one[¹¹C]-radiolabelled conjugate or conjugate-like compound, as definedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, e.g., carriers,diluents, excipients, etc. If formulated as discrete units (e.g.,tablets, etc.), each unit contains a predetermined amount (dosage) ofthe active compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the activeingredient is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the active ingredient in the liquid is from about 1ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1μg/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the conjugate compound, and compositions comprising theconjugate compound, can vary from patient to patient. Determining theoptimal dosage will generally involve the balancing of the level oftherapeutic benefit against any risk or deleterious side effects. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, the severity of the condition, and thespecies, sex, age, weight, condition, general health, and prior medicalhistory of the patient. The amount of compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 mg, 3 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 150 mg, 2 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 200 mg, 2 timesdaily.

However in one embodiment, the conjugate compound is administered to ahuman patient according to the following dosage regime: about 50 orabout 75 mg, 3 or 4 times daily.

In one embodiment, the conjugate compound is administered to a humanpatient according to the following dosage regime: about 100 or about 125mg, 2 times daily.

The dosage amounts described above may apply to the conjugate (includingthe PBD moiety and the linker to the antibody) or to the effectiveamount of PBD compound provided, for example the amount of compound thatis releasable after cleavage of the linker.

For the prevention or treatment of disease, the appropriate dosage of anADC of the invention will depend on the type of disease to be treated,as defined above, the severity and course of the disease, whether themolecule is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The molecule issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. An exemplarydosage of ADC to be administered to a patient is in the range of about0.1 to about 10 mg/kg of patient weight. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs. Anexemplary dosing regimen comprises a course of administering an initialloading dose of about 4 mg/kg, followed by additional doses every week,two weeks, or three weeks of an ADC. Other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein,pertains to that amount of an active compound, or a material,composition or dosage from comprising an active compound, which iseffective for producing some desired prophylactic effect, commensuratewith a reasonable benefit/risk ratio, when administered in accordancewith a desired treatment regimen.

Preparation of Drug Conjugates

Antibody drug conjugates may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including reaction of a nucleophilic group of anantibody with a drug-linker reagent. This method may be employed toprepare the antibody-drug conjugates of the invention.

Nucleophilic groups on antibodies include, but are not limited to sidechain thiol groups, e.g. cysteine. Thiol groups are nucleophilic andcapable of reacting to form covalent bonds with electrophilic groups onlinker moieties such as those of the present invention. Certainantibodies have reducible interchain disulfides, i.e. cysteine bridges.Antibodies may be made reactive for conjugation with linker reagents bytreatment with a reducing agent such as DTT (Cleland's reagent,dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride;Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures,Beverly, Mass.). Each cysteine disulfide bridge will thus form,theoretically, two reactive thiol nucleophiles. Additional nucleophilicgroups can be introduced into antibodies through the reaction of lysineswith 2-iminothiolane (Traut's reagent) resulting in conversion of anamine into a thiol.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, amarsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutan,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus. In one preferred embodiment, the subject/patientis a human.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

In some embodiments, R^(6′), R^(7′), R^(9′), and Y′ are preferably thesame as R⁶, R⁷, R⁹, and Y respectively.

Dimer Link

Y and Y′ are preferably O.

R″ is preferably a C₃₋₇ alkylene group with no substituents. Morepreferably R″ is a C₃, C₅ or C₇ alkylene. Most preferably, R″ is a C₃ orC₅ alkylene.

R⁶ to R⁹

R⁹ is preferably H.

R⁶ is preferably selected from H, OH, OR, SH, NH₂, nitro and halo, andis more preferably H or halo, and most preferably is H.

R⁷ is preferably selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, andhalo, and more preferably independently selected from H, OH and OR,where R is preferably selected from optionally substituted C₁₋₇ alkyl,C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R may be more preferably aC₁₋₄ alkyl group, which may or may not be substituted. A substituent ofinterest is a C₅₋₆ aryl group (e.g. phenyl). Particularly preferredsubstituents at the 7-positions are OMe and OCH₂Ph. Other substituentsof particular interest are dimethylamino (i.e. —NMe₂); —(OC₂H₄)_(q)OMe,where q is from 0 to 2; nitrogen-containing C₆ heterocyclyls, includingmorpholino, piperidinyl and N-methyl-piperazinyl.

These preferences apply to R^(9′), R^(6′) and R^(7′) respectively.

R¹²

When there is a double bond present between C2′ and C3′, R¹² is selectedfrom:

(a) C₅₋₁₀ aryl group, optionally substituted by one or more substituentsselected from the group comprising: halo, nitro, cyano, ether, C₁₋₇alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylene;

(b) C₁₋₅ saturated aliphatic alkyl;

(c) C₃₋₆ saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl.

When R¹² is a C₅₋₁₀ aryl group, it may be a C₅₋₇ aryl group. A C₅₋₇ arylgroup may be a phenyl group or a C₅₋₇ heteroaryl group, for examplefuranyl, thiophenyl and pyridyl. In some embodiments, R¹² is preferablyphenyl. In other embodiments, R¹² is preferably thiophenyl, for example,thiophen-2-yl and thiophen-3-yl.

When R¹² is a C₅₋₁₀ aryl group, it may be a C₈₋₁₀ aryl, for example aquinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl groupmay be bound to the PBD core through any available ring position. Forexample, the quinolinyl may be quinolin-2-yl, quinolin-3-yl,quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl andquinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may bepreferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl,isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yland isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl maybe preferred.

When R¹² is a C₅₋₁₀ aryl group, it may bear any number of substituentgroups. It preferably bears from 1 to 3 substituent groups, with 1 and 2being more preferred, and singly substituted groups being mostpreferred. The substituents may be any position.

Where R¹² is C₅₋₇ aryl group, a single substituent is preferably on aring atom that is not adjacent the bond to the remainder of thecompound, i.e. it is preferably β or γ to the bond to the remainder ofthe compound. Therefore, where the C₅₋₇ aryl group is phenyl, thesubstituent is preferably in the meta- or para-positions, and morepreferably is in the para-position.

Where R¹² is a C₈₋₁₀ aryl group, for example quinolinyl orisoquinolinyl, it may bear any number of substituents at any position ofthe quinoline or isoquinoline rings. In some embodiments, it bears one,two or three substituents, and these may be on either the proximal anddistal rings or both (if more than one substituent).

R¹² Substituents, when R¹² is a C₅₋₁₀ Aryl Group

If a substituent on R¹² when R¹² is a C₅₋₁₀ aryl group is halo, it ispreferably F or Cl, more preferably Cl.

If a substituent on R¹² when R¹² is a C₅₋₁₀ aryl group is ether, it mayin some embodiments be an alkoxy group, for example, a C₁₋₇ alkoxy group(e.g. methoxy, ethoxy) or it may in some embodiments be a C₅₋₇ aryloxygroup (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itselfbe further substituted, for example by an amino group (e.g.dimethylamino).

If a substituent on R¹² when R¹² is a C₅₋₁₀ aryl group is C₁₋₇ alkyl, itmay preferably be a C₁₋₄ alkyl group (e.g. methyl, ethyl, propryl,butyl).

If a substituent on R¹² when R¹² is a C₅₋₁₀ aryl group is C₃₋₇heterocyclyl, it may in some embodiments be C₆ nitrogen containingheterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl,piperazinyl. These groups may be bound to the rest of the PBD moiety viathe nitrogen atom. These groups may be further substituted, for example,by C₁₋₄ alkyl groups.

If the C₆ nitrogen containing heterocyclyl group is piperazinyl, thesaid further substituent may be on the second nitrogen ring atom.

If a substituent on R¹² when R¹² is a C₅₋₁₀ aryl group is bis-oxy-C₁₋₃alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.

If a substituent on R¹² when R¹² is a C₅₋₁₀ aryl group is ester, this ispreferably methyl ester or ethyl ester.

Particularly preferred substituents when R¹² is a C₅₋₁₀ aryl groupinclude methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene,methyl-piperazinyl, morpholino and methylthiophenyl. Other particularlypreferred substituent for R¹² are dimethylaminopropyloxy and carboxy.

Particularly preferred substituted R¹² groups when R¹² is a C₅₋₁₀ arylgroup include, but are not limited to, 4-methoxy-phenyl,3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl,4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl,4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl,isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl,methoxynaphthyl, and naphthyl. Another possible substituted R¹² group is4-nitrophenyl. R¹² groups of particular interest include4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.

When R¹² is C₁₋₅ saturated aliphatic alkyl, it may be methyl, ethyl,propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl orpropyl (n-pentyl or isopropyl). In some of these embodiments, it may bemethyl. In other embodiments, it may be butyl or pentyl, which may belinear or branched.

When R¹² is C₃₋₆ saturated cycloalkyl, it may be cyclopropyl,cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may becyclopropyl.

When R¹² is

each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5. In someembodiments, the total number of carbon atoms in the R¹² group is nomore than 4 or no more than 3.

In some embodiments, one of R²¹, R²² and R²³ is H, with the other twogroups being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In other embodiments, two of R²¹, R²² and R²³ are H, with the othergroup being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In some embodiments, the groups that are not H are selected from methyland ethyl. In some of these embodiments, the groups that re not H aremethyl.

In some embodiments, R²¹ is H.

In some embodiments, R²² is H.

In some embodiments, R²³ is H.

In some embodiments, R²¹ and R²² are H.

In some embodiments, R²¹ and R²³ are H.

In some embodiments, R²² and R²³ are H.

An R¹² group of particular interest is:

When R¹² is

one of R^(25a) and R^(25b) is H and the other is selected from: phenyl,which phenyl is optionally substituted by a group selected from halo,methyl, methoxy; pyridyl; and thiophenyl. In some embodiments, the groupwhich is not H is optionally substituted phenyl. If the phenyl optionalsubstituent is halo, it is preferably fluoro. In some embodiment, thephenyl group is unsubstituted.

When R¹² is

R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl. Ifthe phenyl optional substituent is halo, it is preferably fluoro. Insome embodiment, the phenyl group is unsubstituted.

In some embodiments, R²⁴ is selected from H, methyl, ethyl, ethenyl andethynyl. In some of these embodiments, R²⁴ is selected from H andmethyl.

When there is a single bond present between C2′ and C3′,

R¹² is R

where R^(26a) and R^(26b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(26a) and R^(26b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester.

In some embodiments, it is preferred that R^(26a) and R^(26b) are bothH.

In other embodiments, it is preferred that R^(26a) and R^(26b) are bothmethyl.

In further embodiments, it is preferred that one of R^(26a) and R^(26b)is H, and the other is selected from C₁₋₄ saturated alkyl, C₂₋₃ alkenyl,which alkyl and alkenyl groups are optionally substituted. In thesefurther embodiment, it may be further preferred that the group which isnot H is selected from methyl and ethyl.

R²

The above preferences for R¹² apply equally to R².

R²²

In some embodiments, R²² is of formula IIa.

A in R²² when it is of formula IIa may be phenyl group or a C₅₋₇heteroaryl group, for example furanyl, thiophenyl and pyridyl. In someembodiments, A is preferably phenyl.

Q²-X may be on any of the available ring atoms of the C₅₋₇ aryl group,but is preferably on a ring atom that is not adjacent the bond to theremainder of the compound, i.e. it is preferably β or γ to the bond tothe remainder of the compound. Therefore, where the C₅₋₇ aryl group (A)is phenyl, the substituent (Q²-X) is preferably in the meta- orpara-positions, and more preferably is in the para-position.

In some embodiments, Q¹ is a single bond. In these embodiments, Q² isselected from a single bond and —Z—(CH₂)_(n)—, where Z is selected froma single bond, O, S and NH and is from 1 to 3. In some of theseembodiments, Q² is a single bond. In other embodiments, Q² is—Z—(CH₂)_(n)—. In these embodiments, Z may be O or S and n may be 1 or nmay be 2. In other of these embodiments, Z may be a single bond and nmay be 1.

In other embodiments, Q¹ is —CH═CH—.

In other embodiments, R²² is of formula IIb. In these embodiments,R^(C1), R^(C2) and R^(C3) are independently selected from H andunsubstituted C₁₋₂ alkyl. In some preferred embodiments, R^(C1), R^(C2)and R^(C3) are all H. In other embodiments, R^(C1), R^(C2) and R^(C3)are all methyl. In certain embodiments, R^(C1), R^(C2) and R^(C3) areindependently selected from H and methyl.

X is a group selected from the list comprising: O—R^(L2′), S—R^(L2′),CO₂—R^(L2′), CO—R^(L2′), NH—C(═O)—R^(L2′), NHNH—R^(L2′), CONHNH—R^(L2′),

NR^(N)R^(L2′), wherein R^(N) is selected from the group comprising H andC₁₋₄ alkyl. X may preferably be: OH, SH, CO₂H, —N═C═O or NHR^(N), andmay more preferably be: O—R^(L2′), S—R^(L2′), CO₂—R^(L2′),—NH—C(═O)—R^(L2′) or NH—R^(L2′). Particularly preferred groups include:O—R^(L2′), S—R^(L2′) and NH—R^(L2′), with NH—R^(L2′) being the mostpreferred group.

In some embodiments R²² is of formula IIc. In these embodiments, it ispreferred that Q is NR^(N)—R^(L2′). In other embodiments, Q isO—R^(L2′). In further embodiments, Q is S—R^(L2′). R^(N) is preferablyselected from H and methyl. In some embodiment, R^(N) is H. In otherembodiments, R^(N) is methyl.

In some embodiments, R²² may be -A-CH₂—X and -A-X. In these embodiments,X may be O—R^(L2′), S—R^(L2′), CO₂—R^(L2′), CO—R^(L2′) and NH—R^(L2′).In particularly preferred embodiments, X may be NH—R^(L2).

R¹⁰, R¹¹

In some embodiments, R¹⁰ and R¹¹ together form a double bond between thenitrogen and carbon atoms to which they are bound.

In some embodiments, R¹¹ is OH.

In some embodiments, R¹¹ is OMe.

In some embodiments, R¹¹ is SO_(z)M, where z is 2 or 3 and M is amonovalent pharmaceutically acceptable cation.

R^(11a)

In some embodiments, R^(11a) is OH.

In some embodiments, R^(11a) is OMe.

In some embodiments, R^(11a) is SO_(z)M, where z is 2 or 3 and M is amonovalent pharmaceutically acceptable cation.

R²⁰, R²¹

In some embodiments, R²⁰ and R²¹ together form a double bond between thenitrogen and carbon atoms to which they are bound.

In some embodiments R²⁰ is H.

In some embodiments, R²⁰ is R^(C).

In some embodiments, R²¹ is OH.

In some embodiments, R²¹ is OMe.

In some embodiments, R²¹ is SO_(z)M, where z is 2 or 3 and M is amonovalent pharmaceutically acceptable cation.

R³⁰, R³¹

In some embodiments, R³⁰ and R³¹ together form a double bond between thenitrogen and carbon atoms to which they are bound.

In some embodiments, R³¹ is OH.

In some embodiments, R³¹ is OMe.

In some embodiments, R³¹ is SO_(Z)M, where z is 2 or 3 and M is amonovalent pharmaceutically acceptable cation.

M and z

It is preferred that M is a monovalent pharmaceutically acceptablecation, and is more preferably Na⁺.

z is preferably 3.

Preferred conjugates of the first aspect of the present invention mayhave a D^(L) of formula Ia:

whereR^(L1′), R²⁰ and R²¹ are as defined above;n is 1 or 3;R^(1a) is methyl or phenyl; andR^(2a) is selected from:

Preferred conjugates of the first aspect of the present invention mayhave a D^(L) of formula Ib:

whereR^(L1′), R²⁰ and R²¹ are as defined above;n is 1 or 3; andR^(1a) is methyl or phenyl.

Preferred conjugates of the first aspect of the present invention mayhave a D^(L) of formula Ic:

where R^(L2′), R¹⁰, R¹¹, R³⁰ and R³¹ are as defined aboven is 1 or 3;R^(12a) is selected from:

the amino group is at either the meta or para positions of the phenylgroup.

Preferred conjugates of the first aspect of the present invention mayhave a D^(L) of formula Id:

where R^(L2′), R¹⁰, R¹, R³⁰ and R³¹ are as defined aboven is 1 or 3;R^(1a) is methyl or phenyl;R^(12a) is selected from:

Preferred conjugates of the first aspect of the present invention mayhave a D^(L) of formula Ie:

where R^(L2′), R¹⁰, R¹, R³⁰ and R³¹ are as defined aboven is 1 or 3;R^(1a) is methyl or phenyl;R^(12a) is selected from:

EXAMPLES

General Experimental Methods

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40-60° C.

General LC/MS Conditions:

Method 1 (Default Method, Used Unless Stated Otherwise)

The HPLC (Waters Alliance 2695) was run using a mobile phase of water(A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).Gradient: initial composition 5% B held over 1.0 min, then increase from5% B to 95% B over a 3 min period. The composition was held for 0.1 minat 95% B, then returned to 5% B in 0.03 minutes and hold there for 0.87min. Total gradient run time equals 5 minutes.

Method 2

The HPLC (Waters Alliance 2695) was run using a mobile phase of water(A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).Gradient: initial composition 5% B held over 1.0 minute, then increasefrom 5% B to 95% B over a 2.5 minute period. The composition was heldfor 0.5 minutes at 95% B, then returned to 5% B in 0.1 minutes and holdthere for 0.9 min. Total gradient run time equals 5 minutes.

For Both Methods

Flow rate 3.0 mL/min, 400 μL was split via a zero dead volume tee piecewhich passes into the mass spectrometer. Wavelength detection range: 220to 400 nm. Function type: diode array (535 scans). Column: PhenomenexOnyx Monolithic C18 50×4.60 mm.

The reverse phase flash purification conditions were as follows: TheFlash purification system (Varian 971-Fp) was run using a mobile phaseof water (A) and acetonitrile (B). Gradient: initial composition 5% Bover 20 C.V. (Column Volume) then 5% B to 70% B within 60 C.V. Thecomposition was held for 15 C.V. at 95% B, and then returned to 5% B in5 C.V. and held at 5% B for 10 C.V. Total gradient run time equals 120C.V. Flow rate 6.0 mL/min. Wavelength detection range: 254 nm. Column:Agilent AX1372-1 SF10-5.5gC8.

Preparative HPLC: Reverse-phase ultra-high-performance liquidchromatography (UPLC) was carried out on Phenomenex Gemini NX 5μ C-18columns of the following dimensions: 150×4.6 mm for analysis, and150×21.20 mm for preparative work. All UPLC experiments were performedwith gradient conditions. Eluents used were solvent A (H₂O with 0.1%Formic acid) and solvent B (CH₃CN with 0.1% Formic acid). Flow ratesused were 1.0 ml/min for analytical, and 20.0 ml/min for preparativeHPLC. Detection was at 254 and 280 nm.

Synthesis of Intermediate 12

(a) 1′,3′-Bis[2-methoxy-4-(methoxycarbonyl)phenoxy]propane (3)

Diisopropyl azodicarboxylate (71.3 mL, 73.2 g, 362 mmol) was addeddrop-wise over a period of 60 min to an overhead stirred solution ofmethyl vanillate 2 (60.0 g, 329 mmol) and Ph₃P (129.4 g, 494 mmol) inanhydrous THF (800 mL) at 0-5° C. (ice/acetone) under a nitrogenatmosphere. The reaction mixture was allowed to stir at 0-5° C. for anadditional 1 hour after which time a solution of 1,3-propanediol (11.4mL, 12.0 g, 158 mmol) in THF (12 mL) was added drop-wise over a periodof 20 min. The reaction mixture was allowed to warm to room temperatureand stirred for 5 days. The resulting white precipitate 3 was collectedby vacuum filtration, washed with THF and dried in a vacuum desiccatorto constant weight. Yield=54.7 g (84% based on 1,3-propanediol). Puritysatisfactory by LC/MS (3.20 min (ES+) m/z (relative intensity) 427([M+Na]⁺., 10); ¹H NMR (400 MHz, CDCl₃) δ 7.64 (dd, 2H, J=1.8, 8.3 Hz),7.54 (d, 2H, J=1.8 Hz), 6.93 (d, 2H, J=8.5 Hz), 4.30 (t, 4H, J=6.1 Hz),3.90 (s, 6H), 3.89 (s, 6H), 2.40 (p, 2H, J=6.0 Hz).

(b) 1′,3′-Bis[2-methoxy-4-(methoxycarbonyl)-5-nitrophenoxy]propane (4)

Solid Cu(NO₃)₂.3H₂O (81.5 g, 337.5 mmol) was added slowly to an overheadstirred slurry of the bis-ester 3 (54.7 g, 135 mmol) in acetic anhydride(650 mL) at 0-5° C. (ice/acetone). The reaction mixture was allowed tostir for 1 hour at 0-5° C. and then allowed to warm to room temperature.A mild exotherm (ca. 40-50° C.), accompanied by thickening of themixture and evolution of NO₂ was observed at this stage. Additionalacetic anhydride (300 mL) was added and the reaction mixture was allowedto stir for 16 hours at room temperature. The reaction mixture waspoured on to ice (˜1.5 L), stirred and allowed to return to roomtemperature. The resulting yellow precipitate was collected by vacuumfiltration and dried in a desiccator to afford the desired bis-nitrocompound 4 as a yellow solid. Yield=66.7 g (100%). Purity satisfactoryby LC/MS (3.25 min (ES+) m/z (relative intensity) 517 ([M+Na]⁺., 40); ¹HNMR (400 MHz, CDCl₃) δ 7.49 (s, 2H), 7.06 (s, 2H), 4.32 (t, 4H, J=6.0Hz), 3.95 (s, 6H), 3.90 (s, 6H), 2.45-2.40 (m, 2H).

(c) 1′,3′-Bis(4-carboxy-2-methoxy-5-nitrophenoxy) propane (5)

A slurry of the methyl ester 4 (66.7 g, 135 mmol) in THF (700 mL) wastreated with 1N NaOH (700 mL) and the reaction mixture was allowed tostir vigorously at room temperature. After 4 days stirring, the slurrybecame a dark coloured solution which was subjected to rotaryevaporation under reduced pressure to remove THF. The resulting aqueousresidue was acidified to pH 1 with concentrated HCl and the colourlessprecipitate 5 was collected and dried thoroughly in a vacuum oven (50°C.). Yield=54.5 g (87%). Purity satisfactory by LC/MS (2.65 min (ES+)m/z (relative intensity) 489 ([M+Na]⁺., 30)); ¹H NMR (400 MHz, DMSO-d₆)δ 7.62 (s, 2H), 7.30 (s, 2H), 4.29 (t, 4H, J=6.0 Hz), 3.85 (s, 6H),2.30-2.26 (m, 2H).

(d)1,1′-[[(Propane-1,3-diyl)dioxy]bis[(5-methoxy-2-nitro-1,4-phenylene)carbonyl]]bis[(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate](6)

Oxalyl chloride (24.5 mL, 35.6 g, 281 mmol) was added to a stirredsuspension of the nitrobenzoic acid 5 (43 g, 92.3 mmol) and DMF (6 mL)in anhydrous DCM (600 mL). Following initial effervescence the reactionsuspension became a solution and the mixture was allowed to stir at roomtemperature for 16 hours. Conversion to the acid chloride was confirmedby treating a sample of the reaction mixture with MeOH and the resultingbis-methyl ester was observed by LC/MS. The majority of solvent wasremoved by evaporation under reduced pressure; the resultingconcentrated solution was re-dissolved in a minimum amount of dry DCMand triturated with diethyl ether. The resulting yellow precipitate wascollected by filtration, washed with cold diethyl ether and dried for 1hour in a vacuum oven at 40° C. The solid acid chloride was addedportionwise over a period of 25 min to a stirred suspension of(2S,4R)-methyl-4-hydroxypyrrolidine-2-carboxylate hydrochloride (38.1 g,210 mmol) and TEA (64.5 mL, g, 463 mmol) in DCM (400 mL) at −40° C. (dryice/CH₃CN). Immediately, the reaction was complete as judged by LC/MS(2.47 min (ES+) m/z (relative intensity) 721 ([M+H]⁺., 100). The mixturewas diluted with DCM (200 mL) and washed with 1N HCl (300 mL), saturatedNaHCO₃ (300 mL), brine (400 mL), dried (MgSO₄), filtered and the solventevaporated in vacuo to give the pure product 6 as an orange solid (66.7g, 100%). [o]²² _(D)=−46.1° (c=0.47, CHCl₃); ¹H NMR (400 MHz, CDCl₃)(rotamers) δ 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28(m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H,J=11.1 Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H); ¹³C NMR (100 MHz,CDCl₃) (rotamers) δ 172.4, 166.7, 154.6, 148.4, 137.2, 127.0, 109.7,108.2, 69.7, 65.1, 57.4, 57.0, 56.7, 52.4, 37.8, 29.0; IR (ATR, CHCl₃)3410 (br), 3010, 2953, 1741, 1622, 1577, 1519, 1455, 1429, 1334, 1274,1211, 1177, 1072, 1050, 1008, 871 cm⁻¹; MS (ES⁺) m/z (relativeintensity) 721 ([M+H]⁺., 47), 388 (80); HRMS [M+H]⁺. theoreticalC₃₁H₃₆N₄O₁₆ m/z 721.2199. found (ES⁺) m/z 721.2227.

(e)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(hydroxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](7)

Method A:

A solution of the nitro-ester 6 (44 g, 61.1 mmol) in MeOH (2.8 L) wasadded to freshly purchased Raney® nickel (˜50 g of a ˜50% slurry in H₂O)and anti-bumping granules in a 5 L 3-neck round bottomed flask. Themixture was heated at reflux and then treated dropwise with a solutionof hydrazine hydrate (21.6 mL, 22.2 g, 693 mmol) in MeOH (200 mL) atwhich point vigorous effervescence was observed. When the addition wascomplete (˜45 min) additional Raney® nickel was added carefully untileffervescence had ceased and the initial yellow colour of the reactionmixture was discharged. The mixture was heated at reflux for a further 5min at which point the reaction was deemed complete by TLC (90:10 v/vCHCl₃/MeOH) and LC/MS (2.12 min (ES+) m/z (relative intensity) 597([M+H]⁺., 100)). The reaction mixture was filtered hot immediatelythrough a sinter funnel containing celite with vacuum suction. Thefiltrate was reduced in volume by evaporation in vacuo at which point acolourless precipitate formed which was collected by filtration anddried in a vacuum desiccator to provide 7 (31 g, 85%). [α]²⁷ _(D)=+404°(c=0.10, DMF); ¹H NMR (400 MHz, DMSO-d₆) δ 10.2 (s, 2H, NH), 7.26 (s,2H), 6.73 (s, 2H), 5.11 (d, 2H, J=3.98 Hz, OH), 4.32-4.27 (m, 2H),4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H, J=12.1, 3.60 Hz), 3.43(dd, 2H, J=12.0, 4.72 Hz), 2.67-2.57 (m, 2H), 2.26 (p, 2H, J=5.90 Hz),1.99-1.89 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.1, 164.0, 149.9,144.5, 129.8, 117.1, 111.3, 104.5, 54.8, 54.4, 53.1, 33.5, 27.5; IR(ATR, neat) 3438, 1680, 1654, 1610, 1605, 1516, 1490, 1434, 1379, 1263,1234, 1216, 1177, 1156, 1115, 1089, 1038, 1018, 952, 870 cm⁻¹; MS (ES⁺)m/z (relative intensity) 619 ([M+Na]⁺., 10), 597 ([M+H]⁺., 52), 445(12), 326 (11); HRMS [M+H]⁺. theoretical C₂₉H₃₂N₄O₁₀ m/z 597.2191. found(ES⁺) m/z 597.2205.

Method B:

A suspension of 10% Pd/C (7.5 g, 10% w/w) in DMF (40 mL) was added to asolution of the nitro-ester 6 (75 g, 104 mmol) in DMF (360 mL). Thesuspension was hydrogenated in a Parr hydrogenation apparatus over 8hours. Progress of the reaction was monitored by LC/MS after thehydrogen uptake had stopped. Solid Pd/C was removed by filtration andthe filtrate was concentrated by rotary evaporation under vacuum (below10 mbar) at 40° C. to afford a dark oil containing traces of DMF andresidual charcoal. The residue was digested in EtOH (500 mL) at 40° C.on a water bath (rotary evaporator bath) and the resulting suspensionwas filtered through celite and washed with ethanol (500 mL) to give aclear filtrate. Hydrazine hydrate (10 mL, 321 mmol) was added to thesolution and the reaction mixture was heated at reflux. After 20 minutesthe formation of a white precipitate was observed and reflux was allowedto continue for a further 30 minutes. The mixture was allowed to cooldown to room temperature and the precipitate was retrieved byfiltration, washed with diethyl ether (2:1 volume of precipitate) anddried in a vacuum desiccator to provide 7 (50 g, 81%). Analytical datafor method B: Identical to those obtained for Method A (opticalrotation, ¹H NMR, LC/MS and TLC).

(f)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](8)

TBSCl (27.6 g, 182.9 mmol) and imidazole (29.9 g, 438.8 mmol) were addedto a cloudy solution of the tetralactam 7 (21.8 g, 36.6 mmol) inanhydrous DMF (400 mL) at 0° C. (ice/acetone). The mixture was allowedto stir under a nitrogen atmosphere for 3 hours after which time thereaction was deemed complete as judged by LC/MS (3.90 min (ES+) m/z(relative intensity) 825 ([M+H]⁺., 100). The reaction mixture was pouredonto ice (˜1.75 L) and allowed to warm to room temperature withstirring. The resulting white precipitate was collected by vacuumfiltration, washed with H₂O, diethyl ether and dried in the vacuumdesicator to provide pure 8 (30.1 g, 99%). [α]²³ _(D)=+234° (c=0.41,CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54(s, 2H), 4.50 (p, 2H, J=5.38 Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H),3.73-3.63 (m, 4H), 2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m,2H), 0.86 (s, 18H), 0.08 (s, 12H); ¹³C NMR (100 MHz, CDCl₃) δ 170.4,165.7, 151.4, 146.6, 129.7, 118.9, 112.8, 105.3, 69.2, 65.4, 56.3, 55.7,54.2, 35.2, 28.7, 25.7, 18.0, −4.82 and −4.86; IR (ATR, CHCl₃) 3235,2955, 2926, 2855, 1698, 1695, 1603, 1518, 1491, 1446, 1380, 1356, 1251,1220, 1120, 1099, 1033 cm⁻¹; MS (ES⁺) m/z (relative intensity) 825([M+H]⁺., 62), 721 (14), 440 (38); HRMS [M+H]⁺. theoreticalC₄₁H₆₀N₄O₁₀Si₂ m/z 825.3921. found (ES⁺) m/z 825.3948.

(g)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-(tert-butyldimethylsilyloxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](9)

A solution of n-BuLi (68.3 mL of a 1.6 M solution in hexane, 109 mmol)was added dropwise to a stirred suspension of the tetralactam 8 (30.08g, 36.4 mmol) in anhydrous THF (600 mL) at −30° C. (dry ice/ethyleneglycol) under a nitrogen atmosphere. The reaction mixture was allowed tostir at this temperature for 1 hour (now a reddish orange colour) atwhich point a solution of SEMCl (19.3 mL, 18.2 g, 109 mmol) in anhydrousTHF (120 mL) was added dropwise. The reaction mixture was allowed toslowly warm to room temperature and was stirred for 16 hours under anitrogen atmosphere. The reaction was deemed complete as judged by TLC(EtOAc) and LC/MS (4.77 min (ES+) m/z (relative intensity) 1085([M+H]⁺., 100). The THF was removed by evaporation in vacuo and theresulting residue dissolved in EtOAc (750 mL), washed with H₂O (250 mL),brine (250 mL), dried (MgSO₄) filtered and evaporated in vacuo toprovide the crude N10-SEM-protected tetralactam 9 as an oil (max^(m)39.5 g, 100%). Product carried through to next step withoutpurification. [α]²³ _(D)=+1630 (c=0.41, CHCl₃); ¹H NMR (400 MHz, CDCl₃)δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98 Hz), 4.68 (d, 2H,J=9.99 Hz), 4.57 (p, 2H, J=5.77 Hz), 4.29-4.19 (m, 6H), 3.89 (s, 6H),3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81 Hz), 2.03-1.90(m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H); ¹³C NMR (100MHz, CDCl₃) δ 170.0, 165.7, 151.2, 147.5, 133.8, 121.8, 111.6, 106.9,78.1, 69.6, 67.1, 65.5, 56.6, 56.3, 53.7, 35.6, 30.0, 25.8, 18.4, 18.1,−1.24, −4.73; IR (ATR, CHCl₃) 2951, 1685, 1640, 1606, 1517, 1462, 1433,1360, 1247, 1127, 1065 cm⁻¹; MS (ES⁺) m/z (relative intensity) 1113([M+Na]⁺., 48), 1085 ([M+H]⁺., 100), 1009 (5), 813 (6); HRMS [M+H]⁺.theoretical C₅₃H₈₈N₄O₁₂Si₄ m/z 1085.5548. found (ES⁺) m/z 1085.5542.

(h)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS,2R)-2-hydroxy-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](10)

A solution of TBAF (150 mL of a 1.0 M solution in THF, 150 mmol) wasadded to a stirred solution of the crude bis-silyl ether 9 [84.0 g(max^(m) 56.8 g), 52.4 mmol] in THF (800 mL) at room temperature. Afterstirring for 1 hour, analysis of the reaction mixture by TLC (95:5 v/vCHCl₃/MeOH) revealed completion of reaction. The THF was removed byevaporation under reduced pressure at room temperature and the resultingresidue dissolved in EtOAc (500 mL) and washed with NH₄Cl (300 mL). Thecombined organic layers were washed with brine (60 mL), dried (MgSO₄),filtered and evaporated under reduced pressure to provide the crudeproduct. Purification by flash chromatography (gradient elution: 100%CHCl₃ to 96:4 v/v CHCl₃/MeOH) gave the pure tetralactam 10 as a whitefoam (36.0 g, 79%). LC/MS 3.33 min (ES+) m/z (relative intensity) 879([M+Na]⁺., 100), 857 ([M+H]⁺., 40); [α]²³ _(D)=+2020 (c=0.34, CHCl₃); ¹HNMR (400 MHz, CDCl₃) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0Hz), 4.72 (d, 2H, J=10.0 Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83Hz), 4.20-4.16 (m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (brs, 2H, OH), 2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77 Hz), 2.11-2.05 (m,2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 169.5,165.9, 151.3, 147.4, 133.7, 121.5, 111.6, 106.9, 79.4, 69.3, 67.2, 65.2,56.5, 56.2, 54.1, 35.2, 29.1, 18.4, −1.23; IR (ATR, CHCl₃) 2956, 1684,1625, 1604, 1518, 1464, 1434, 1361, 1238, 1058, 1021 cm⁻¹; MS (ES⁺) m/z(relative intensity) 885 ([M+29]⁺., 70), 857 ([M+H]⁺., 100), 711 (8),448 (17); HRMS [M+H]⁺. theoretical C₄₁H₆₀N₄O₁₂Si₂ m/z 857.3819. found(ES⁺) m/z 857.3826.

(i)1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](11)

Diol 10 (25.6 g, 30 mmol, 1 eq.), NaOAc (6.9 g, 84 mmol, 2.8 eq.) andTEMPO (188 mg, 1.2 mmol, 0.04 eq.) were dissolved in DCM (326 mL) underAr. This was cooled to −8° C. (internal temperature) and TCCA (9.7 g, 42mmol, 1.4 eq.) was added portionwise over 15 minutes. TLC (EtOAc) andLC/MS [3.60 min. (ES+) m/z (relative intensity) 854.21 ([M+H]⁺., 40),(ES−) m/z (relative intensity) 887.07 ([M−H+Cl]⁻., 10)] after 30 minutesindicated that reaction was complete. Cold DCM (200 mL) was added andthe mixture was filtered through a pad of Celite before washing with asolution of saturated sodium hydrogen carbonate/sodium thiosulfate (1:1v/v; 200 mL×2). The organic layer was dried with MgSO₄, filtered and thesolvent removed in vacuo to yield a yellow/orange sponge (25.4 g, 99%).LC/MS [3.60 min. (ES+) m/z (relative intensity) 854.21 ([M+H]⁺., 40);[α]²⁰ _(D)=+291° (c=0.26, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.32 (s,2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1 Hz), 4.75 (d, 2H, J=10.1 Hz),4.60 (dd, 2H, J=9.85, 3.07 Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H),3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85 Hz), 2.76 (dd, 2H, J=19.2,9.90 Hz), 2.42 (p, 2H, J=5.77 Hz), 0.98-0.91 (m, 4H), 0.00 (s, 18H); ¹³CNMR (100 MHz, CDCl₃) δ 206.8, 168.8, 165.9, 151.8, 148.0, 133.9, 120.9,111.6, 107.2, 78.2, 67.3, 65.6, 56.3, 54.9, 52.4, 37.4, 29.0, 18.4,−1.24; IR (ATR, CHCl₃) 2957, 1763, 1685, 1644, 1606, 1516, 1457, 1434,1360, 1247, 1209, 1098, 1066, 1023 cm⁻¹; MS (ES⁺) m/z (relativeintensity) 881 ([M+29]⁺., 38), 853 ([M+H]⁺., 100), 707 (8), 542 (12);HRMS [M+H]⁺. theoretical C₄₁H₅₆N₄O₁₂Si₂ m/z 853.3506. found (ES⁺) m/z853.3502.

(j) 1,1′-[[(Propane-1,3-diyl)dioxy]bis(11aS)-7-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]-10-((2-(trimethylsilyl)ethoxy)methyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]-benzodiazepin-5,11-dione](12)

Anhydrous 2,6-lutidine (5.15 mL, 4.74 g, 44.2 mmol) was injected in oneportion to a vigorously stirred solution of bis-ketone 11 (6.08 g, 7.1mmol) in dry DCM (180 mL) at −45° C. (dry ice/acetonitrile) under anitrogen atmosphere. Anhydrous triflic anhydride, taken from a freshlyopened ampoule (7.2 mL, 12.08 g, 42.8 mmol), was injected rapidlydropwise, while maintaining the temperature at −40° C. or below. Thereaction mixture was allowed to stir at −45° C. for 1 hour at whichpoint TLC (50/50 v/v n-hexane/EtOAc) revealed the complete consumptionof starting material. The cold reaction mixture was immediately dilutedwith DCM (200 mL) and, with vigorous shaking, washed with water (1×100mL), 5% citric acid solution (1×200 mL) saturated NaHCO₃ (200 mL), brine(100 mL) and dried (MgSO₄). Filtration and evaporation of the solventunder reduced pressure afforded the crude product which was purified byflash column chromatography (gradient elution: 90:10 v/v n-hexane/EtOActo 70:30 v/v n-hexane/EtOAc) to afford bis-enol triflate 12 as a yellowfoam (5.5 g, 70%). LC/MS 4.32 min (ES+) m/z (relative intensity) 1139([M+Na]⁺., 20); [α]²⁴ _(D)=+271° (c=0.18, CHCl₃); ¹H NMR (400 MHz,CDCl₃) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97 Hz), 5.51 (d,2H, J=10.1 Hz), 4.76 (d, 2H, J=10.1 Hz), 4.62 (dd, 2H, J=11.0, 3.69 Hz),4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H,J=16.3, 11.0, 2.36 Hz), 2.43 (p, 2H, J=5.85 Hz), 1.23-0.92 (m, 4H), 0.02(s, 18H); ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 162.7, 151.9, 148.0, 138.4,133.6, 120.2, 118.8, 111.9, 107.4, 78.6, 67.5, 65.6, 56.7, 56.3, 30.8,29.0, 18.4, −1.25; IR (ATR, CHCl₃) 2958, 1690, 1646, 1605, 1517, 1456,1428, 1360, 1327, 1207, 1136, 1096, 1060, 1022, 938, 913 cm⁻¹; MS (ES⁺⁾m/z (relative intensity) 1144 ([M+28]⁺., 100), 1117 ([M+H]⁺., 48), 1041(40), 578 (8); HRMS [M+H]⁺. theoretical C₄₃H₅₄N₄O₁₆Si₂S₂F₆ m/z1117.2491. found (ES⁺) m/z 1117.2465.

Example 1

(a)(S)-8-(3-(((S)-2-(4-aminophenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yltrifluoromethanesulfonate (13)

Pd(PPh₃)₄ (116.9 mg, 0.101 mmol) was added to a stirred mixture of thebis-enol triflate 12 (5.65 g, 5.06 mmol), 4-Aminophenylboronic acidpinacol ester (1 g, 4.56 mmol), Na₂CO₃ (2.46 g, 23.2 mmol), MeOH (37mL), toluene (74 mL) and water (37 mL). The reaction mixture was allowedto stir at 30° C. under a nitrogen atmosphere for 24 hours after whichtime all the boronic ester has consumed. The reaction mixture was thenevaporated to dryness before the residue was taken up in EtOAc (150 mL)and washed with H₂O (2×100 mL), brine (150 mL), dried (MgSO₄), filteredand evaporated under reduced pressure to provide the crude product.Purification by flash chromatography (gradient elution: 80:20 v/vHexane/EtOAc to 60:40 v/v Hexane/EtOAc) afforded product 13 as ayellowish foam (2.4 g, 45%). LC/MS 4.02 min (ES+) m/z (relativeintensity) 1060.21 ([M+H]⁺., 100); ¹H-NMR: (CDCl₃, 400 MHz) δ 7.40 (s,1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J=8.5 Hz), 7.15 (t, 1H,J=2.0 Hz), 6.66 (d, 2H, J=8.5 Hz), 5.52 (d, 2H, J=10.0 Hz), 4.77 (d, 1H,J=10.0 Hz), 4.76 (d, 1H, J=10.0 Hz), 4.62 (dd, 1H, J=3.7, 11.0 Hz), 4.58(dd, 1H, J=3.4, 10.6 Hz), 4.29 (t, 4H, J=5.6 Hz), 4.00-3.85 (m, 8H),3.80-3.60 (m, 4H), 3.16 (ddd, 1H, J=2.4, 11.0, 16.3 Hz), 3.11 (ddd, 1H,J=2.2, 10.5, 16.1 Hz), 2.43 (p, 2H, J=5.9 Hz), 1.1-0.9 (m, 4H), 0.2 (s,18H). ¹³C-NMR: (CDCl₃, 100 MHz) δ 169.8, 168.3, 164.0, 162.7, 153.3,152.6, 149.28, 149.0, 147.6, 139.6, 134.8, 134.5, 127.9, 127.5, 125.1,123.21, 121.5, 120.5, 120.1, 116.4, 113.2, 108.7, 79.8, 79.6, 68.7,68.5, 67.0, 66.8, 58.8, 58.0, 57.6, 32.8, 32.0, 30.3, 19.7, 0.25.

(b)(S)-2-(4-Aminophenyl)-8-(3-(((S)-2-cyclopropyl-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(1H,11aH)-dione(14)

Triphenylarsine (0.24 g, 0.8 mmol), silver (I) oxide (1.02 g, 4.4 mmol),cyclopropylboronic acid (0.47 g, 5.5 mmol) and starting material 13(1.15 g, 1.1 mmol) were dissolved in dioxane (30 mL) under an argonatmosphere. Potassium phosphate tribasic (2.8 g, 13.2 mmol) wasground-up with a pestle and mortar and quickly added to the reactionmixture. The reaction mixture was evacuated and flushed with argon 3times and heated to 71° C. Palladium(II)bis(benzonitrile chloride) (84mg, 0.22 mmol) was added and the reaction vessel was evacuated andflushed with argon 3 times. After 10 minutes a small sample was takenfor analysis by TLC (80:20 v/v ethyl acetate/hexane) and LC/MS. After 30minutes the reaction had gone to completion (LC/MS analysis indicatedcomplete consumption of starting material) and the reaction was filteredthrough celite and the filter pad washed with ethyl acetate (400 mL).The filtrate was washed with water (2×200 mL) and brine (2×200 mL). Theorganic layer was dried with MgSO₄, filtered and the solvent removed invacuo. Purification by silica gel column chromatography (30:70 v/vHexane/Ethyl acetate) afforded the product 14 as an orangey/yellow solid(0.66 g, 63%). Method 1, LC/MS (3.85 min (ES⁺) m/z (relative intensity)952.17 ([M+H]⁺., 100). ¹H NMR (400 MHz, CDCl₃) δ 7.36 (d, 2H, J=8.4 Hz),7.30 (s, 1H), 7.25-7.19 (m, 4H), 6.68 (s, 1H), 6.62 (d, 2H, J=8.4 Hz),5.49 (dd, 2H, J=5.6, 10.0 Hz), 4.73 (app. t, 2H, J=10.8 Hz), 4.54 (dd,1H, J=3.2, 10.4 Hz), 4.40 (dd, 1H, J=3.2, 10.4 Hz), 4.29-4.23 (m, 4H),3.91-3.85 (m, 7H), 3.80-3.71 (m, 2H), 3.70-3.61 (m, 2H), 3.38-3.32 (m,1H), 3.12-3.01 (m, 1H), 2.50-2.69 (m, 1H), 2.40 (q, 2H, J=5.6 Hz),1.50-1.43 (m, 1H), 0.99-0.71 (m, 6H), 0.54-0.59 (m, 2H), 0.00 (s, 18H)ppm.

(c)(S)-2-(4-Aminophenyl)-8-(3-(((S)-2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one(15)

SEM dilactam 14 (0.66 g, 0.69 mmol) was dissolved in THF (23 mL) andcooled to −78° C. under an argon atmosphere. Super-Hydride® solution(1.7 mL, 1 M in THF) was added drop wise over 5 minutes while monitoringthe temperature. After 20 minutes a small sample was taken and washedwith water for LC/MS analysis. Water (50 mL) was added and the cold bathwas removed. The organic layer was extracted and washed with brine (60mL). The combined aqueous layers were washed with CH₂Cl₂/MeOH (90/10v/v) (2×50 mL). The combined organic layers were dried with MgSO₄,filtered and the solvent removed in vacuo. The crude product wasdissolved in MeOH (48 mL), CH₂Cl₂ (18 mL) and water (6 mL) andsufficient silica gel was added to afford a thick suspension. After 5days stirring, the suspension was filtered through a sintered funnel andwashed with CH₂Cl₂/MeOH (9:1) (˜200 mL) until product ceased to beeluted. The organic layer was washed with brine (2×70 mL), dried withMgSO₄, filtered and the solvent removed in vacuo. Purification by silicagel column chromatography (100% CHCl₃ to 96/4 v/v CHCl₃/MeOH) affordedthe product 15 as a yellow solid (302 mg, 66%). Method 1, LC/MS (2.42min (ES⁺) m/z (relative intensity) 660.74 ([M+H]⁺., 30). ¹H NMR (400MHz, CDCl₃) δ 7.86 (d, 1H, J=3.6 Hz), 7.78 (d, 1H, J=3.6 Hz), 7.58-7.44(m, 3H), 7.34-7.20 (m, 3H), 6.88-6.66 (m, 4H), 4.35-4.15 (m, 6H),3.95-3.75 (m, 7H), 3.39-3.22 (m, 1H), 3.14-3.04 (m, 1H), 2.93-2.85 (m,1H), 2.46-2.36 (m, 2H), 1.49-1.41 (m, 1H), 0.80-0.72 (m, 2H), 0.58-0.51(app. s, 2H) ppm.

(d)Allyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(16)

In a degassed round bottom flask filled with argon, HO-Ala-Val-alloc(149.6 mg, 0.549 mmol) and EEDQ (135.8 mg, 0.549 mmol) were dissolved ina 9:1 mixture of dry CH₂Cl₂/MeOH (5 mL). The flask was wrapped inaluminium foil and the reaction mixture was allowed to stir at roomtemperature for 1 hour before starting material 15 (302 mg, 0.457 mmol)was added. The reaction mixture was left to stir for a further 40 hoursat room temperature before the volatiles were removed by rotaryevaporation under reduced pressure (the reaction was followed by LC/MS,RT starting material 2.32 min, (ES+660.29 ([M+H]⁺., 100)). The crudeproduct was directly purified by silica gel chromatography column (100%CHCl₃ to 90/10 v/v CHCl₃/MeOH) to afford the pure product (16) in 42%yield (174 mg). Method 2 LC/MS (2.70 min (ES+) m/z (relative intensity)914.73 ([M+H]⁺., 60), 660.43 (60), 184.31 (100)).

(e)(2S)-2-amino-N-((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(17)

The starting material 16 (170 mg, 0.185 mmol) was dissolved in dryCH₂Cl₂ (5 mL) in a round bottom flask filled with argon, beforepyrrolidine (41 μL, 0.21 mmol) was added. The flask was purged/refilledthree times with argon before Pd(PPh₃)₄ (14 mg, 0.084 mmol) was addedand the flushing operation repeated. After 1 hour, complete consumptionof starting material was observed (the reaction was followed by LC/MS)and Et₂O (50 mL) was added to the reaction mixture which was allowed tostir until all the product had crashed out of solution. The solid wasfiltered through a sintered funnel and washed twice with Et₂O (2×25 mL).The collecting flask was replaced and the isolated solid was dissolvedin CHCl₃ (100 mL or until all the product had passed through thesintered funnel). The volatiles were then removed by rotary evaporationunder reduced pressure to afford the crude product 17 which was useddirectly in the next step (168 mg). LC/MS method 2 (2.70 min (ES+) m/z(relative intensity) 830.27 ([M+H]⁺., 50), 660.13 (80), 171.15 (100)).

(f)N—((R)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide(18)

Starting material 17 (154 mg, 0.185 mmol) and EDCl.HCl (110 mg, 0.185mmol) were solubilised in dry CH₂Cl₂ (5 mL) in a round bottom flaskpurged and filled with argon. The mixture was left to stir at roomtemperature for 1 hour before PEG₈-maleimide (35.6 mg, 0.185 mmol) wasadded and the reaction mixture stirred for a further 16 hours (or untilthe reaction is complete, monitored by LC/MS). The reaction solution wasdiluted with CH₂Cl₂ (50 mL) and the organics were washed with H₂O (50mL) and brine (50 mL) before being dried with MgSO₄, filtered and thesolvent removed by rotary evaporation under reduced pressure to affordthe crude product. Purification on silica gel column chromatography(100% CHCl₃ to 85/15 v/v CHCl₃/MeOH) gave the desired product (135 mg),however remaining traces of unreacted PEG₈-maleimide were observed (byLC/MS, 2.21 min, method 2). Automated reverse phase silica gelchromatography (H₂O/CH₃CN) (see general information for conditions)successfully removed the impurity affording pure final product (18, 37mg of pure product starting from 110 mg, 33%). Overall yield=17%. Method2 LC/MS (2.58 min (ES+) m/z (relative intensity) 1404.03 ([M+H]⁺., 20),702.63 (100)). ¹H NMR (400 MHz, CDCl₃) δ 7.91 (t, J=3.5 Hz, 1H), 7.80(d, J=4.0 Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H),7.54-7.50 (m, 2H), 7.45 (s, 1H), 7.39-7.31 (m, 2H), 6.87 (d, J=10.5 Hz,2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m,7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42(dd, J=10.2, 5.2 Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J=16.1, 4.1 Hz,1H), 2.62-2.49 (m, 4H), 2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s,1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J=9.2, 5.3 Hz, 2H),0.57 (dd, J=9.2, 5.3 Hz, 2H), NH were not observed.

Example 2

(a)(R)-2-((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanoic acid (20b)

HO-Ala-Val-H 20a (350 mg, 1.86 mmol) and Na₂CO₃ (493 mg, 4.65 mmol) weredissolved in distilled H₂O (15 mL) and the mixture was cooled to 0° C.before dioxane (15 mL) was added (partial precipitation of the aminoacid salt occurred). A solution of Fmoc-Cl (504 mg, 1.95 mmol) indioxane (15 mL) was added dropwise with vigorous stirring over 10minutes. The resulting mixture was stirred at 0° C. for 2 hours beforethe ice bath was removed and stirring was maintained for 16 hours. Thesolvent was removed by rotary evaporation under reduced pressure and theresidue dissolved in water (150 mL). The pH was adjusted from 9 to 2with 1N HCl and the aqueous layer was subsequently extracted with EtOAc(3×100 mL). The combined organics were washed with brine (100 mL), driedwith MgSO₄, filtered and the volatiles removed by rotary evaporationunder reduced pressure to afford pure HO-Ala-Val-Fmoc 20b (746 mg, 97%yield). LC/MS 2.85 min (ES+) m/z (relative intensity) 410.60; ¹H-NMR(400 MHz, CDCl₃) δ 7.79 (d, J=7.77 Hz, 2H), 7.60 (d, J=7.77 Hz, 2H),7.43 (d, J=7.5 Hz, 2H), 7.34 (d, J=7.5 Hz, 2H), 6.30 (bs, 1H), 5.30 (bs,1H), 4.71-7.56 (m, 1H), 4.54-4.36 (m, 2H), 4.08-3.91 (m, 1H), 2.21-2.07(m, 1H), 1.50 (d, J=7.1 Hz, 3H), 1.06-0.90 (m, 6H).

(b) (9H-fluoren-9-yl)methyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)propan-2-yl)amino)butan-2-yl)carbamate(20)

4-Aminophenylboronic acid pinacol ester was added (146.9 mg, 0.67 mmol)was added to a solution of HO-Ala-Val-Fmoc 20b (330 mg, 0.8 mmol), DCC(166 mg, 0.8 mmol) and DMAP (5 mg, cat.) in dry DCM (8 mL) previouslystirred for 30 minutes at room temperature in a flask flushed withargon. The reaction mixture was then allowed to stir at room temperatureovernight. The reaction was followed by LCMS and TLC. The reactionmixture was diluted with CH₂Cl₂ and the organics were washed with H₂Oand brine before being dried with MgSO₄, filtered and the solventremoved by rotary evaporation under reduced pressure. The crude productwas dryloaded on a silica gel chromatography column (Hexane/EtOAc, 6:4)and pure product 20 was isolated as a white solid in 88% yield (360 mg).

(c)8-(3-((2-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)phenyl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yltrifluoromethanesulfonate (21)

Bis-triflate 12 (2.03 g, 1.81 mmol), boronic pinacol ester (1 g, 1.63mmol) and Na₂CO₃ (881 mg, 8.31 mmol) were dissolved in a mixture oftoluene/MeOH/H₂O, 2:1:1 (40 mL). The reaction flask was purged andfilled with argon three times beforetetrakis(triphenylphosphine)palladium(0) (41 mg, 0.035 mmol) was addedand the reaction mixture heated to 30° C. overnight. The solvents wereremoved under reduce pressure and the residue was taken up in H₂O (100mL) and extracted with EtOAc (3×100 mL). The combined organics werewashed with brine (100 mL), dried with MgSO₄, filtered and the volatilesremoved by rotary evaporation under reduced pressure. The crude productwas purified by silica gel chromatography column (Hexane/EtOAc, 8:2 to25:75) to afford pure 21 in 33% yield (885 mg). LC/MS 3.85 min (ES+) m/z(relative intensity) 1452.90; ¹H NMR (400 MHz, CDCl₃) δ 7.78-7.16 (m,17H), 7.13 (s, 1H), 6.51-6.24 (m, 1H), 5.51 (dd, J=10.0, 5.1 Hz, 2H),5.36-5.11 (m, 1H), 4.74 (dd, J=10.1, 4.4 Hz, 2H), 4.70-4.53 (m, 2H),4.47 (d, J=6.4 Hz, 1H), 4.37 (d, J=7.2 Hz, 1H), 4.27 (m, 4H), 4.20-4.14(m, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.77 (ddd, J=16.7, 9.0, 6.4 Hz,3H), 3.71-3.61 (m, 2H), 3.24-2.91 (m, 3H), 2.55-2.33 (m, 2H), 2.22-2.07(m, 1H), 1.52-1.37 (m, 3H), 1.04-0.86 (m, 10H), 0.00 (s, 18H).

(d)(9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (22)

Triphenylarsine (42 mg, 0.137 mmol) was added to a mixture ofPBD-triflate 21 (250 mg, 0.172 mmol), cyclopropylboronic acid (73.9 mg,0.86 mmol), silver oxide (159 mg, 0.688 mmol) and potassium phosphatetribasic (438 mg, 2.06 mmol) in dry dioxane (10 mL) under an argonatmosphere. The reaction was flushed with argon 3 times andbis(benzonitrile)palladium(II) chloride (13.2 mg, 0.034 mmol) was added.The reaction was flushed with Argon 3 more times before being warmed to75° C. and stirred for 10 minutes. The reaction mixture was filteredthrough a pad of celite which was subsequently rinsed with ethylacetate. The solvent was removed by rotary evaporation under reducedpressure. The resulting residue was subjected to flash columnchromatography (silica gel; 1% methanol/chloroform). Pure fractions werecollected and combined, and excess eluent was removed by rotaryevaporation under reduced pressure to afford the desired product 22 (132mg, 50% yield). LC/MS 3.83 min (ES+) m/z (relative intensity) 1345.91;¹H NMR (400 MHz, CDCl₃) δ 7.88-7.14 (m, 17H), 6.69 (s, 1H), 6.45-6.25(m, 1H), 5.57-5.41 (m, 2H), 5.34-5.14 (m, 1H), 4.78-4.67 (m, 2H),4.62-4.55 (m, 1H), 4.50-4.45 (m, 2H), 4.51-4.44 (m, 1H), 4.31-4.21 (m,4H), 4.16 (m, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.82-3.71 (m, 2H), 3.66(m, 3H), 3.40-3.28 (m, 1H), 3.07 (m, 1H), 2.70-2.57 (m, 1H), 2.47-2.36(m, 2H), 2.15 (m, 1H), 1.51-1.40 (m, 3H), 1.03-0.87 (m, 11H), 0.77-0.71(m, 2H), 0.60-0.54 (m, 2H), 0.00 (t, J=3.0 Hz, 18H).

(e)(9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(23)

A solution of Super-Hydride® (0.5 mL, 1M in THF) was added dropwise to asolution of SEM dilactam 22 (265 mg g, 0.19 mmol) in THF (10 mL) at −78°C. under an argon atmosphere. The addition was completed over 5 minutesin order to maintain the internal temperature of the reaction mixtureconstant. After 20 minutes, an aliquot was quenched with water for LC/MSanalysis, which revealed that the reaction was complete. Water (20 mL)was added to the reaction mixture and the cold bath was removed. Theorganic layer was extracted with EtOAc (3×30 mL) and the combinedorganics were washed with brine (50 mL), dried with MgSO₄, filtered andthe solvent removed by rotary evaporation under reduced pressure. Thecrude product was dissolved in MeOH (12 mL), CH₂Cl₂ (6 mL), water (2 mL)and enough silica gel to form a thick stirring suspension. After 5 days,the suspension was filtered through a sintered funnel and washed withCH₂Cl₂/MeOH (9:1) (200 mL) until the elution of the product wascomplete. The organic layer was washed with brine (2×70 mL), dried withMgSO₄, filtered and the solvent removed by rotary evaporation underreduced pressure. Purification by silica gel column chromatography (100%CHCl₃ to 96% CHCl₃/4% MeOH) afforded the product 23 as a yellow solid(162 mg, 78%). LC/MS 3.02 min (ES+) m/z (relative intensity) 1052.37.

(f)(2S)-2-amino-N-((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(17)

Excess piperidine was added (0.2 mL, 2 mmol) to a solution ofSEM-dilactam 23 (76 mg, 0.073 mmol) in DMF (1 mL). The mixture wasallowed to stir at room temperature for 20 min, at which point thereaction had gone to completion (as monitored by LC/MS). The reactionmixture was diluted with CH₂Cl₂ (75 mL) and the organic phase was washedwith H₂O (3×75 mL) until complete piperidine removal. The organic phasewas dried over MgSO₄, filtered and excess solvent removed by rotaryevaporation under reduced pressure to afford crude product 17 which wasused as such in the next step. LC/MS 2.32 min (ES+) m/z (relativeintensity) 830.00.

(g)N-((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide(18)

EDCl hydrochloride (14 mg, 0.0732 mmol) was added to a suspension ofMaleimide-PEG₈-acid (43.4 mg, 0.0732 mmol) in dry CH₂Cl₂ (5 mL) underargon atmosphere. The mixture was stirred for 1 hour at room temperaturebefore PBD 17 (60.7 mg, 0.0732 mmol) was added. Stirring was maintaineduntil the reaction was complete (usually 5 hours). The reaction wasdiluted with CH₂Cl₂ and the organic phase was washed with H₂O and brinebefore being dried over MgSO₄, filtered and excess solvent removed byrotary evaporation under reduced pressure by rotary evaporation underreduced pressure. The product was purified by careful silica gelchromatography (slow elution starting with 100% CHCl₃ up to 9:1CHCl₃/MeOH) followed by reverse phase chromatography to remove unreactedmaleimide-PEG₈-acid. The product 18 was isolated in 17.6% (21.8 mg).LC/MS 2.57 min (ES+) m/z (relative intensity) 1405.30; ¹H NMR (400 MHz,CDCl₃) 7.91 (t, J=3.5 Hz, 1H), 7.80 (d, J=4.0 Hz, 1H), 7.75 (d, J=8.8Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H),7.39-7.31 (m, 2H), 6.87 (d, J=10.5 Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m,2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H),3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J=10.2, 5.2 Hz, 2H),3.16-3.07 (m, 1H), 2.92 (dd, J=16.1, 4.1 Hz, 1H), 2.62-2.49 (m, 4H),2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H),1.10-0.93 (m, 6H), 0.79 (dd, J=9.2, 5.3 Hz, 2H), 0.57 (dd, J=9.2, 5.3Hz, 2H), NH were not observed.

Example 3

(a)(S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yltrifluoromethanesulfonate (24)

Pd(PPh₃)₄ (20.6 mg, 0.018 mmol) was added to a stirred mixture of thebis-enol triflate 12 (500 mg, 0.44 mmol), N-methyl piperazine boronicester (100 mg, 0.4 mmol), Na₂CO₃ (218 mg, 2.05 mmol), MeOH (2.5 mL),toluene (5 mL) and water (2.5 mL). The reaction mixture was allowed tostir at 30° C. under a nitrogen atmosphere for 24 hours after which timeall the boronic ester has consumed. The reaction mixture was thenevaporated to dryness before the residue was taken up in EtOAc (100 mL)and washed with H₂O (2×50 mL), brine (50 mL), dried (MgSO₄), filteredand evaporated under reduced pressure to provide the crude product.Purification by flash chromatography (gradient elution: 80:20 v/vHexane/EtOAc to 60:40 v/v Hexane/EtOAc) afforded product 24 as ayellowish foam (122.6 mg, 25%). LC/MS 3.15 min (ES+) m/z (relativeintensity) 1144 ([M+H]⁺., 20%).

(b) (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (25)

PBD-triflate 24 (359 mg, 0.314 mmol), boronic pinacol ester 20 (250 mg,0.408 mmol) and triethylamine (0.35 mL, 2.51 mmol) were dissolved in amixture of toluene/MeOH/H₂O, 2:1:1 (3 mL). The microwave vessel waspurged and filled with argon three times beforetetrakis(triphenylphosphine)palladium(0) (21.7 mg, 0.018 mmol) was addedand the reaction mixture placed in the microwave at 80° C. for 10minutes. Subsequently, CH₂Cl₂ (100 mL) was added and the organics werewashed with water (2×50 mL) and brine (50 mL) before being dried withMgSO₄, filtered and the volatiles removed by rotary evaporation underreduced pressure. The crude product was purified by silica gelchromatography column (CHCl₃/MeOH, 100% to 9:1) to afford pure 25 (200mg, 43% yield). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478([M+H]⁺., 100%).

(c) (9H-fluoren-9-yl) methyl((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(26)

A solution of Super-Hydride® (0.34 mL, 1M in THF) was added dropwise toa solution of SEM-dilactam 25 (200 mg, 0.135 mmol) in THF (5 mL) at −78°C. under an argon atmosphere. The addition was completed over 5 minutesin order to maintain the internal temperature of the reaction mixtureconstant. After 20 minutes, an aliquot was quenched with water for LC/MSanalysis, which revealed that the reaction was complete. Water (20 mL)was added to the reaction mixture and the cold bath was removed. Theorganic layer was extracted with EtOAc (3×30 mL) and the combinedorganics were washed with brine (50 mL), dried with MgSO₄, filtered andthe solvent removed by rotary evaporation under reduced pressure. Thecrude product was dissolved in MeOH (6 mL), CH₂Cl₂ (3 mL), water (1 mL)and enough silica gel to form a thick stirring suspension. After 5 days,the suspension was filtered through a sintered funnel and washed withCH₂Cl₂/MeOH (9:1) (100 mL) until the elution of the product wascomplete. The organic layer was washed with brine (2×50 mL), dried withMgSO₄, filtered and the solvent removed by rotary evaporation underreduced pressure. Purification by silica gel column chromatography (100%CHCl₃ to 96% CHCl₃/4% MeOH) afforded the product 26 as a yellow solid(100 mg, 63%). LC/MS 2.67 min (ES+) m/z (relative intensity) 1186([M+H]⁺., 5%).

(d)(S)-2-amino-N—((S)-1-((4-((R)-7-methoxy-8-(3-(((R)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(27)

Excess piperidine was added (0.1 mL, 1 mmol) to a solution of PBD 26(36.4 mg, 0.03 mmol) in DMF (0.9 mL). The mixture was allowed to stir atroom temperature for 20 min, at which point the reaction had gone tocompletion (as monitored by LC/MS). The reaction mixture was dilutedwith CH₂Cl₂ (50 mL) and the organic phase was washed with H₂O (3×50 mL)until complete piperidine removal. The organic phase was dried overMgSO₄, filtered and excess solvent removed by rotary evaporation underreduced pressure to afford crude product 27 which was used as such inthe next step. LC/MS 2.20 min (ES+) m/z (relative intensity) 964([M+H]⁺., 5%).

(e) 6-(2,5-dioxo-2,5-dihydro1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-((4-((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(28)

EDCl hydrochloride (4.7 mg, 0.03 mmol) was added to a suspension of6-maleimidohexanoic acid (6.5 mg, 0.03 mmol) in dry CH₂Cl₂ (3 mL) underargon atmosphere. The mixture was stirred for 1 hour at room temperaturebefore PBD 27 (34 mg, crude) was added. Stirring was maintained untilthe reaction was complete (6 hours). The reaction was diluted withCH₂Cl₂ and the organic phase was washed with H₂O and brine before beingdried over MgSO₄, filtered and excess solvent removed by rotaryevaporation under reduced pressure by rotary evaporation under reducedpressure. The product was purified by careful silica gel chromatography(slow elution starting with 100% CHCl₃ up to 9:1 CHCl₃/MeOH) followed byreverse phase chromatography to remove unreacted maleimide-PEG₈-acid.The product 28 was isolated in 41% over two steps (14.6 mg). LC/MS 2.40min (ES+) m/z (relative intensity) 1157 ([M+H]⁺., 5%)

Example 4—Alternative Synthesis of Compound 25

PBD-triflate 21 (469 mg, 0.323 mmol), boronic pinacol ester (146.5 mg,0.484 mmol) and Na₂CO₃ (157 mg, 1.48 mmol) were dissolved in a mixtureof toluene/MeOH/H₂O, 2:1:1 (10 mL). The reaction flask was purged withargon three times before tetrakis(triphenylphosphine)palladium(0) (7.41mg, 0.0064 mmol) was added and the reaction mixture heated to 30° C.overnight. The solvents were removed under reduced pressure and theresidue was taken up in H₂O (50 mL) and extracted with EtOAc (3×50 mL).The combined organics were washed with brine (100 mL), dried with MgSO₄,filtered and the volatiles removed by rotary evaporation under reducedpressure. The crude product was purified by silica gel columnchromatography (CHCl₃ 100% to CHCl₃/MeOH 95%:5%) to afford pure 25 in33% yield (885 mg). LC/MS 3.27 min (ES+) m/z (relative intensity) 1478([M+H]⁺., 100%).

Example 5

(a)(S)-2-(4-Aminophenyl)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,1-c][1,4]benzodiazepine-5,11(10H,11aH)-dione(29)

3,4-(Methylenedioxy)phenyl boronic acid (356 mg, 2.1 mmol, 1.3 equiv.),TEA (1.8 mL, 12.9 mmol, 8 equiv.) and triflate/aniline 13 (1.75 g, 1.7mmol, 1 equiv.) were dissolved in a mixture of ethanol (7 mL), toluene(13 mL) and water (2 mL) under an Ar atmosphere. The reaction mixturewas evacuated and flushed with Ar 3 times, before addition oftetrakis(triphenylphosphine)palladium(0) (114 mg, 0.1 mmol, 0.06equiv.). The flask was again evacuated and flushed with Ar 3 times andheated in a microwave at 80° C. for 8 minutes with 30 secondspre-stirring time. Analysis by TLC (80:20 v/v ethyl acetate/hexane)indicated complete consumption of starting material. The reactionmixture was diluted with dichloromethane (50 mL) and washed with water(50 mL). The organic layer was dried with MgSO₄, filtered and thesolvent removed in vacuo. Purification by silica gel columnchromatography (60:40 to 20:80 v/v hexane/ethyl acetate) afforded theproduct 29 as a yellow solid (1.21 g, 71%). LC/MS (3.92 min (ES⁺) m/z(relative intensity) 1032.44 ([M+H]⁺., 100).

(b)(S)-2-(4-Aminophenyl)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one(30)

SEM dilactam 29 (0.25 g, 0.24 mmol, 1 equiv.) was dissolved in THF (8mL) and cooled to −78° C. under an Ar atmosphere. Super-Hydride® (0.6mL, 1 M in THF, 2.5 equiv.) was added drop wise over 5 minutes whilemonitoring the temperature. After 20 minutes a small sample was takenand worked-up for LCMS analysis. Water (50 mL) was added, the cold bathwas removed and the solution washed with ethyl acetate (50 mL). Theorganic layer was extracted and washed with brine (60 mL), dried withMgSO₄, filtered and the solvent removed in vacuo. The crude product wasdissolved in EtOH (15 mL), CH₂Cl₂ (7.5 mL) and water (2.5 mL) and enoughsilica gel was added until it was a thick suspension. After 5 daysstirring, it was filtered through a sintered funnel and washed withCH₂Cl₂/MeOH (9:1) (100 mL) until product ceased to be eluted. Theorganic layer was washed with brine (2×50 mL), dried with MgSO₄,filtered and the solvent removed in vacuo. Purification by silica gelcolumn chromatography (CHCl₃ with 1% to 4% MeOH gradient) afforded theproduct 30 as a yellow solid (94 mg, 53%). LC/MS (2.53 min (ES⁺) m/z(relative intensity) 739.64 ([M]+, 70).

(c) Allyl((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(31)

Under an Ar atmosphere, Alanine-Valine-Alloc (180 mg, 0.66 mmol, 1.2equiv.) was stirred with EEDQ (163 mg, 0.66 mmol, 1.2 equiv.) inanhydrous CH₂Cl₂(21 mL) and methanol (1 mL) for 1 hour. The PBD 30 (407mg, 0.55 mmol, 1 equiv.) was dissolved in anhydrous CH₂Cl₂ (21 mL) andmethanol (1 mL) and added to the reaction. LC/MS after 5 days stirringat room temperature showed majority product formation. The solvent wasremoved in vacuo before purification by column chromatography (CH₂Cl₂with 1% to 6% MeOH gradient) to yield the product 31 as a yellow solid(184 mg, 34%). LC/MS (2.95 min (ES⁺) m/z (relative intensity) 994.95([M+H]⁺., 60).

(d)(S)-2-Amino-N—((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(32)

The imine 31 (100 mg, 0.1 mmol, 1 equiv.) was dissolved in anhydrous DCM(10 mL) (with the aid of one drop of methanol to aid dissolution) underan Ar atmosphere. Pyrrolidine (30 μL, 0.15 mmol, 1.5 equiv.) was addeddrop wise before the flask was evacuated and flushed with Ar threetimes. Pd(PPh₃)₄ (7 mg, 6 μmol, 0.06 equiv.) was added and the flask wasevacuated and flushed with Ar three times. LC/MS analysis after 1 hourindicated product formation and complete loss of starting material. Et₂O(60 mL) was added to the reaction mixture and it was left to stir untilall the product had crashed out of solution. The precipitate wasfiltered through a sintered funnel and washed twice with Et₂O (2×20 mL).The collection flask was replaced and the isolated solid was dissolvedand washed through the sinter with CHCl₃ (100 mL). The solvent wasremoved in vacuo to afford the crude product 32 as a yellow solid whichwas used directly in the next step. LC/MS (1.14 min (ES⁺) m/z (relativeintensity) 910.40 ([M+H]⁺., 67).

(e)N—((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(Benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide(33)

The imine 32 (92 mg, 0.1 mmol, 1.1 equiv.) was dissolved in CHCl₃ (6 mL)with one drop of anhydrous MeOH to aid dissolution. Maleimide-PEG₈-acid(53 mg, 0.09 mmol, 1 equiv.) was added followed by EEDQ (33 mg, 0.14mmol, 1.5 equiv.). This was left to stir vigorously at room temperatureunder Ar for 4 days until LC/MS analysis showed majority productformation. The solvent was removed in vacuo and the crude product waspartially purified by silica gel column chromatography (CHCl3 with 1% to10% MeOH gradient) yielding 33 (81 mg). The material was purifiedfurther by preparative HPLC to give 33 as a yellow solid (26.3 mg, 18%).Fast Formic run: LC/MS (1.39 min (ES+) m/z (relative intensity) 1485.00([M+H]+., 64).

Example 6

(a) 9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (34)

The triflate 21 (0.5 g, 0.35 mmol, 1 equiv.), 3,4-(methylenedioxy)phenylboronic acid (75 mg, 0.45 mmol, 1.3 equiv.) and Na₂CO₃ (0.17 g, 1.6mmol, 4.5 equiv.) were dissolved in toluene (11 mL), EtOH (5.5 mL) andwater (5.5 mL) under an Ar atmosphere. The flask was evacuated andflushed with Ar three times. Pd(PPh₃)₄ (24 mg, 0.02 mmol, 0.06 equiv.)was added and again the flask was evacuated and flushed with Ar threetimes. This was heated to 30° C. and left stirring overnight. Analysisby LC/MS showed complete loss of starting material. The solvent wasremoved in vacuo and the residue dissolved in water (60 mL) beforewashing with ethyl acetate (60 mL×3). The combined organic layers werewashed with brine (50 mL), dried with MgSO₄, filtered and the solventremoved in vacuo. Purification by column chromatography (50:50 to 25:75v/v hexane/ethyl acetate) afforded the product 34 as a yellow solid (310mg, 64%). LC/MS (1.44 min (ES⁻) m/z (relative intensity) 1423.35([M−H]⁻., 79).

(b) (9H-Fluoren-9-yl)methyl((S)-1-(((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (35)

SEM dilactam 34 (0.31 g, 0.22 mmol, 1 equiv.) was dissolved in THF (10mL) and cooled to −78° C. under an Ar atmosphere. Super-Hydride® (0.5mL, 1 M in THF, 2.5 equiv.) was added drop wise over 5 minutes whilemonitoring the temperature. After 30 minutes a small sample was takenand worked-up for LC/MS analysis. Water (50 mL) was added, the cold bathwas removed and the solution washed with ethyl acetate (50 mL). Theorganic layer was extracted and washed with brine (60 mL), dried withMgSO₄, filtered and the solvent removed in vacuo. The crude product wasdissolved in EtOH (13.2 mL), CH₂Cl₂ (6.6 mL) and water (2.2 mL) andenough silica gel was added until it was a thick suspension. After 5days stirring, it was filtered through a sintered funnel and washed withCH₂Cl₂/MeOH (9:1) (100 mL) until product ceased to be eluted. Theorganic layer was washed with brine (2×50 mL), dried with MgSO₄,filtered and the solvent removed in vacuo. Purification by silica gelcolumn chromatography (CHCl₃ with 1% to 4% MeOH gradient) afforded thepure product 35 as a yellow solid (185 mg, 75%). LC/MS (1.70 min (ES⁺)m/z (relative intensity) 1132.85 ([M+H]⁺., 60).

(c)(S)-2-Amino-N—((S)-1-((4-((S)-8-(3-(((S)-2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-7-methoxy-5-oxo-5,11a-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(32)

The imine 35 (82 mg, 0.07 mmol, 1 equiv.) was dissolved in DMF (1 mL)before piperidine (0.2 mL, 2 mmol, excess) was added slowly. Thissolution was left to stir at room temperature for 20 minutes until LC/MSanalysis showed complete consumption of starting material. The reactionmixture was diluted with CH₂Cl₂ (50 mL), washed with water (50 mL x 4),dried with MgSO₄, filtered and the solvent removed in vacuo. The product33 was used without further purification in the next step. LC/MS (1.15min (ES⁺) m/z (relative intensity) 910.60 ([M+H]⁺., 58).

Example 7 (i)(S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone(49)

(a) 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzaldehyde (42)

Neat triisopropylsilylchloride (56.4 mL, 262 mmol) was added to amixture of imidazole (48.7 g, 715.23 mmol) and4-hydroxy-5-methoxy-2-nitrobenzaldehyde 41 (47 g, 238 mmol) (groundtogether). The mixture was heated until the phenol and imidazole meltedand went into solution (100° C.). The reaction mixture was allowed tostir for 15 minutes and was then allowed to cool, whereupon a solid wasobserved to form at the bottom of the flask (imidazole chloride). Thereaction mixture was diluted with 5% EtOAc/hexanes and loaded directlyonto silica gel and the pad was eluted with 5% EtOAc/hexanes, followedby 10% EtOAc/hexanes (due to the low excess, very little unreactedTIPSCl was found in the product). The desired product was eluted with 5%ethyl acetate in hexane. Excess eluent was removed by rotary evaporationunder reduced pressure, followed by drying under high vacuum to afford acrystalline light sensitive solid (74.4 g, 88%). Purity satisfactory byLC/MS (4.22 min (ES+) m/z (relative intensity) 353.88 ([M+H]⁺., 100));¹H NMR (400 MHz, CDCl₃) δ 10.43 (s, 1H), 7.60 (s, 1H), 7.40 (s, 1H),3.96 (s, 3H), 1.35-1.24 (m, 3H), 1.10 (m, 18H).

(b) 5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoic acid (43)

A solution of sodium chlorite (47.3 g, 523 mmol, 80% technical grade)and sodium dihydrogenphosphate monobasic (35.2 g, 293 mmol) (NaH₂PO₄) inwater (800 mL) was added to a solution of compound 2 (74 g, 209 mmol) intetrahydrofuran (500 mL) at room temperature. Hydrogen peroxide (60%w/w, 140 mL, 2.93 mol) was immediately added to the vigorously stirredbiphasic mixture. The reaction mixture evolved gas (oxygen), thestarting material dissolved and the temperature of the reaction mixturerose to 45° C. After 30 minutes LC/MS revealed that the reaction wascomplete. The reaction mixture was cooled in an ice bath andhydrochloric acid (1 M) was added to lower the pH to 3 (this step wasfound unnecessary in many instances, as the pH at the end of thereaction is already acidic; please check the pH before extraction). Thereaction mixture was then extracted with ethyl acetate (1 L) and theorganic phases washed with brine (2×100 mL) and dried over magnesiumsulphate. The organic phase was filtered and excess solvent removed byrotary evaporation under reduced pressure to afford the product 43 inquantitative yield as a yellow solid. LC/MS (3.93 min (ES−) m/z(relative intensity) 367.74 ([M−H]⁻., 100)); ¹H NMR (400 MHz, CDCl₃) δ7.36 (s, 1H), 7.24 (s, 1H), 3.93 (s, 3H), 1.34-1.22 (m, 3H), 1.10 (m,18H).

(c)((2S,4R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-hydroxypyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone(45)

DCC (29.2 g, 141 mmol, 1.2 eq) was added to a solution of acid 3 (43.5g, 117.8 mmol, 1 eq), and hydroxybenzotriazole hydrate (19.8 g, 129.6mmol, 1.1 eq) in dichloromethane (200 mL) at 0° C. The cold bath wasremoved and the reaction was allowed to proceed for 30 mins at roomtemperature, at which time a solution of(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine 44 (30 g,129.6 mmol, 1.1 eq) and triethylamine (24.66 mL, 176 mmol, 1.5 eq) indichloromethane (100 mL) was added rapidly at −10° C. under argon (onlarge scale, the addition time could be shortened by cooling thereaction mixture even further. The reaction mixture was allowed to stirat room temperature for 40 minutes to 1 hour and monitored by LC/MS andTLC (EtOAc). The solids were removed by filtration over celite and theorganic phase was washed with cold aqueous 0.1 M HCl until the pH wasmeasured at 4 or 5. The organic phase was then washed with water,followed by saturated aqueous sodium bicarbonate and brine. The organiclayer was dried over magnesium sulphate, filtered and excess solventremoved by rotary evaporation under reduced pressure. The residue wassubjected to column flash chromatography (silica gel; gradient 40/60ethyl acetate/hexane to 80/20 ethyl acetate/hexane). Excess solvent wasremoved by rotary evaporation under reduced pressure afforded the pureproduct 45, (45.5 g of pure product 66%, and 17 g of slightly impureproduct, 90% in total). LC/MS 4.43 min (ES+) m/z (relative intensity)582.92 ([M+H]⁺., 100); ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s, 1H), 6.74 (s,1H), 4.54 (s, 1H), 4.40 (s, 1H), 4.13 (s, 1H), 3.86 (s, 3H), 3.77 (d,J=9.2 Hz, 1H), 3.36 (dd, J=11.3, 4.5 Hz, 1H), 3.14-3.02 (m, 1H),2.38-2.28 (m, 1H), 2.10 (ddd, J=13.3, 8.4, 2.2 Hz, 1H), 1.36-1.19 (m,3H), 1.15-1.05 (m, 18H), 0.91 (s, 9H), 0.17-0.05 (m, 6H), (presence ofrotamers).

(d)(S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)pyrrolidin-3-one(46)

TCCA (8.82 g, 40 mmol, 0.7 eq) was added to a stirred solution of 45(31.7 g, 54 mmol, 1 eq) and TEMPO (0.85 g, 5.4 mmol, 0.1 eq) in drydichloromethane (250 mL) at 0° C. The reaction mixture was vigorouslystirred for 20 minutes, at which point TLC (50/50 ethyl acetate/hexane)revealed complete consumption of the starting material. The reactionmixture was filtered through celite and the filtrate washed with aqueoussaturated sodium bicarbonate (100 mL), sodium thiosulphate (9 g in 300mL), brine (100 mL) and dried over magnesium sulphate. Rotaryevaporation under reduced pressure afforded product 46 in quantitativeyield. LC/MS 4.52 min (ES+) m/z (relative intensity) 581.08 ([M+H]⁺.,100); ¹H NMR (400 MHz, CDCl₃) δ 7.78-7.60 (m, 1H), 6.85-6.62 (m, 1H),4.94 (dd, J=30.8, 7.8 Hz, 1H), 4.50-4.16 (m, 1H), 3.99-3.82 (m, 3H),3.80-3.34 (m, 3H), 2.92-2.17 (m, 2H), 1.40-1.18 (m, 3H), 1.11 (t, J=6.2Hz, 18H), 0.97-0.75 (m, 9H), 0.15-−0.06 (m, 6H), (presence of rotamers).

(e)(S)-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yltrifluoromethanesulfonate (47)

Triflic anhydride (27.7 mL, 46.4 g, 165 mmol, 3 eq) was injected(temperature controlled) to a vigorously stirred suspension of ketone 46(31.9 g, 55 mmol, 1 eq) in dry dichloromethane (900 mL) in the presenceof 2,6-lutidine (25.6 mL, 23.5 g, 220 mmol, 4 eq, dried over sieves) at−50° C. (acetone/dry ice bath). The reaction mixture was allowed to stirfor 1.5 hours when LC/MS, following a mini work-up(water/dichloromethane), revealed the reaction to be complete. Water wasadded to the still cold reaction mixture and the organic layer wasseparated and washed with saturated sodium bicarbonate, brine andmagnesium sulphate. The organic phase was filtered and excess solventwas removed by rotary evaporation under reduced pressure. The residuewas subjected to column flash chromatography (silica gel; 10/90 v/vethyl acetate/hexane), removal of excess eluent afforded the product 47(37.6 g, 96%) LC/MS, method 2, 4.32 min (ES+) m/z (relative intensity)712.89 ([M+H]⁺., 100); ¹H NMR (400 MHz, CDCl₃) δ 7.71 (s, 1H), 6.75 (s,1H), 6.05 (d, J=1.8 Hz, 1H), 4.78 (dd, J=9.8, 5.5 Hz, 1H), 4.15-3.75 (m,5H), 3.17 (ddd, J=16.2, 10.4, 2.3 Hz, 1H), 2.99 (ddd, J=16.3, 4.0, 1.6Hz, 1H), 1.45-1.19 (m, 3H), 1.15-1.08 (m, 18H), 1.05 (s, 6H), 0.95-0.87(m, 9H), 0.15-0.08 (m, 6H).

(f)(S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone(48)

Triphenylarsine (1.71 g, 5.60 mmol, 0.4 eq) was added to a mixture oftriflate 47 (10.00 g, 14 mmol, 1 eq), methylboronic acid (2.94 g, 49.1mmol, 3.5 eq), silver oxide (13 g, 56 mmol, 4 eq) and potassiumphosphate tribasic (17.8 g, 84 mmol, 6 eq) in dry dioxane (80 mL) underan argon atmosphere. The reaction was flushed with argon 3 times andbis(benzonitrile)palladium(II) chloride (540 mg, 1.40 mmol, 0.1 eq) wasadded. The reaction was flushed with argon 3 more times before beingwarmed instantaneously to 110° C. (the drysyn heating block waspreviously warmed to 110° C. prior addition of the flask). After 10 minsthe reaction was cooled to room temperature and filtered through a padcelite. The solvent was removed by rotary evaporation under reducedpressure. The resulting residue was subjected to column flashchromatography (silica gel; 10% ethyl acetate/hexane). Pure fractionswere collected and combined, and excess eluent was removed by rotaryevaporation under reduced pressure afforded the product 48 (4.5 g, 55%).LC/MS, 4.27 min (ES+) m/z (relative intensity) 579.18 ([M+H]⁺., 100); ¹HNMR (400 MHz, CDCl₃) δ 7.70 (s, 1H), 6.77 (s, 1H), 5.51 (d, J=1.7 Hz,1H), 4.77-4.59 (m, 1H), 3.89 (s, 3H), 2.92-2.65 (m, 1H), 2.55 (d, J=14.8Hz, 1H), 1.62 (d, J=1.1 Hz, 3H), 1.40-1.18 (m, 3H), 1.11 (s, 9H), 1.10(s, 9H), 0.90 (s, 9H), 0.11 (d, J=2.3 Hz, 6H).

(g) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrol-1-yl)methanone(49)

Zinc powder (28 g, 430 mmol, 37 eq) was added to a solution of compound48 (6.7 g, 11.58 mmol) in 5% formic acid in ethanol v/v (70 mL) ataround 15° C. The resulting exotherm was controlled using an ice bath tomaintain the temperature of the reaction mixture below 30° C. After 30minutes the reaction mixture was filtered through a pad of celite. Thefiltrate was diluted with ethyl acetate and the organic phase was washedwith water, saturated aqueous sodium bicarbonate and brine. The organicphase was dried over magnesium sulphate, filtered and excess solventremoved by rotary evaporation under reduced pressure. The resultingresidue was subjected to flash column chromatography (silica gel; 10%ethyl acetate in hexane). The pure fractions were collected and combinedand excess solvent was removed by rotary evaporation under reducedpressure to afford the product 49 (5.1 g, 80%). LC/MS, 4.23 min (ES+)m/z (relative intensity) 550.21 ([M+H]⁺., 100); ¹H NMR (400 MHz, CDCl₃)δ 7.28 (s, 1H), 6.67 (s, 1H), 6.19 (s, 1H), 4.64-4.53 (m, J=4.1 Hz, 1H),4.17 (s, 1H), 3.87 (s, 1H), 3.77-3.69 (m, 1H), 3.66 (s, 3H), 2.71-2.60(m, 1H), 2.53-2.43 (m, 1H), 2.04-1.97 (m, J=11.9 Hz, 1H), 1.62 (s, 3H),1.26-1.13 (m, 3H), 1.08-0.99 (m, 18H), 0.82 (s, 9H), 0.03-−0.03 (m,J=6.2 Hz, 6H).

(ii) (11S,11aS)-allyl11-((tert-butyldimethylsilyl)oxy)-8-((5-iodopentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate

(a)(S)-allyl(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate(50)

Allyl chloroformate (0.30 mL, 3.00 mmol, 1.1 eq) was added to a solutionof amine 49 (1.5 g, 2.73 mmol) in the presence of dry pyridine (0.48 mL,6.00 mmol, 2.2 eq) in dry dichloromethane (20 mL) at −78° C.(acetone/dry ice bath). After 30 minutes, the bath was removed and thereaction mixture was allowed to warm to room temperature. The reactionmixture was diluted with dichloromethane and saturated aqueous coppersulphate was added. The organic layer was then washed sequentially withsaturated aqueous sodium bicarbonate and brine. The organic phase wasdried over magnesium sulphate, filtered and excess solvent removed byrotary evaporation under reduced pressure to afford the product 50 whichwas used directly in the next reaction. LC/MS, 4.45 min (ES+) m/z(relative intensity) 632.91 ([M+H]⁺., 100)

(b)(S)-allyl(2-(2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate(51)

The crude 50 was dissolved in a 7:1:1:2 mixture of aceticacid/methanol/tetrahydrofuran/water (28:4:4:8 mL) and allowed to stir atroom temperature. After 3 hours, complete disappearance of startingmaterial was observed by LC/MS. The reaction mixture was diluted withethyl acetate and washed sequentially with water (2×500 mL), saturatedaqueous sodium bicarbonate (200 mL) and brine. The organic phase wasdried over magnesium sulphate filtered and excess ethyl acetate removedby rotary evaporation under reduced pressure. The resulting residue wassubjected to flash column chromatography (silica gel, 25% ethyl acetatein hexane). Pure fractions were collected and combined and excess eluentwas removed by rotary evaporation under reduced pressure to afford thedesired product 51 (1 g, 71%). LC/MS, 3.70 min (ES+) m/z (relativeintensity) 519.13 ([M+H]⁺., 95); ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H),7.69 (s, 1H), 6.78 (s, 1H), 6.15 (s, 1H), 5.95 (ddt, J=17.2, 10.5, 5.7Hz, 1H), 5.33 (dq, J=17.2, 1.5 Hz, 1H), 5.23 (ddd, J=10.4, 2.6, 1.3 Hz,1H), 4.73 (tt, J=7.8, 4.8 Hz, 1H), 4.63 (dt, J=5.7, 1.4 Hz, 2H), 4.54(s, 1H), 3.89-3.70 (m, 5H), 2.87 (dd, J=16.5, 10.5 Hz, 1H), 2.19 (dd,J=16.8, 4.6 Hz, 1H), 1.70 (d, J=1.3 Hz, 3H), 1.38-1.23 (m, 3H), 1.12 (s,10H), 1.10 (s, 8H).

(c) (11S,11aS)-allyl11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(52)

Dimethyl sulphoxide (0.35 mL, 4.83 mmol, 2.5 eq) was added dropwise to asolution of oxalyl chloride (0.2 mL, 2.32 mmol, 1.2 eq) in drydichloromethane (10 mL) at −78° C. (dry ice/acetone bath) under anatmosphere of argon. After 10 minutes a solution of 51 (1 g, 1.93 mmol)in dry dichloromethane (8 mL) was added slowly with the temperaturestill at −78° C. After 15 min triethylamine (1.35 mL, dried over 4 Åmolecular sieves, 9.65 mmol, 5 eq) was added dropwise and the dryice/acetone bath was removed. The reaction mixture was allowed to reachroom temperature and was extracted with cold hydrochloric acid (0.1 M),saturated aqueous sodium bicarbonate and brine. The organic phase wasdried over magnesium sulphate, filtered and excess dichloromethane wasremoved by rotary evaporation under reduced pressure to afford product52 (658 mg, 66%). LC/MS, 3.52 min (ES+) m/z (relative intensity) 517.14([M+H]⁺., 100); ¹H NMR (400 MHz, CDCl₃) δ 7.20 (s, 1H), 6.75-6.63 (m,J=8.8, 4.0 Hz, 2H), 5.89-5.64 (m, J=9.6, 4.1 Hz, 2H), 5.23-5.03 (m, 2H),4.68-4.38 (m, 2H), 3.84 (s, 3H), 3.83-3.77 (m, 1H), 3.40 (s, 1H),3.05-2.83 (m, 1H), 2.59 (d, J=17.1 Hz, 1H), 1.78 (d, J=1.3 Hz, 3H),1.33-1.16 (m, 3H), 1.09 (d, J=2.2 Hz, 9H), 1.07 (d, J=2.1 Hz, 9H).

(d) (11S,11aS)-allyl11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(53)

Tert-butyldimethylsilyltriflate (0.70 mL, 3.00 mmol, 3 eq) was added toa solution of compound 52 (520 mg, 1.00 mmol) and 2,6-lutidine (0.46 mL,4.00 mmol, 4 eq) in dry dichloromethane (40 mL) at 0° C. under argon.After 10 min, the cold bath was removed and the reaction mixture wasstirred at room temperature for 1 hour. The reaction mixture wasextracted with water, saturated aqueous sodium bicarbonate and brine.The organic phase was dried over magnesium sulphate, filtered and excesswas removed by rotary evaporation under reduced pressure. The resultingresidue was subjected to flash column chromatography (silica gel;gradient, 10% ethyl acetate in hexane to 20% ethyl acetate in hexane).Pure fractions were collected and combined and excess eluent was removedby rotary evaporation under reduced pressure to give the product 53 (540mg, 85%). LC/MS, 4.42 min (ES+) m/z (relative intensity) 653.14([M+Na]⁺., 100); ¹H NMR (400 MHz, CDCl₃) δ 7.20 (s, 1H), 6.71-6.64 (m,J=5.5 Hz, 2H), 5.83 (d, J=9.0 Hz, 1H), 5.80-5.68 (m, J=5.9 Hz, 1H),5.14-5.06 (m, 2H), 4.58 (dd, J=13.2, 5.2 Hz, 1H), 4.36 (dd, J=13.3, 5.5Hz, 1H), 3.84 (s, 3H), 3.71 (td, J=10.1, 3.8 Hz, 1H), 2.91 (dd, J=16.9,10.3 Hz, 1H), 2.36 (d, J=16.8 Hz, 1H), 1.75 (s, 3H), 1.31-1.16 (m, 3H),1.12-1.01 (m, J=7.4, 2.1 Hz, 18H), 0.89-0.81 (m, 9H), 0.25 (s, 3H), 0.19(s, 3H).

(e) (11S,11aS)-allyl11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(54)

Lithium acetate (87 mg, 0.85 mmol) was added to a solution of compound53 (540 mg, 0.85 mmol) in wet dimethylformamide (6 mL, 50:1 DMF/water).After 4 hours, the reaction was complete and the reaction mixture wasdiluted with ethyl acetate (25 mL) and washed with aqueous citric acidsolution (pH˜3), water and brine. The organic layer was dried overmagnesium sulphate filtered and excess ethyl acetate was removed byrotary evaporation under reduced pressure. The resulting residue wassubjected to flash column chromatography (silica gel; gradient, 25% to75% ethyl acetate in hexane). Pure fractions were collected and combinedand excess eluent was removed by rotary evaporation under reducedpressure to give the product 54 (400 mg, quantitative). LC/MS, (3.33 min(ES+) m/z (relative intensity) 475.26 ([M+H]⁺, 100).

(f) (11S,11aS)-allyl11-((tert-butyldimethylsilyl)oxy)-8-((5-iodopentyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(55)

Diiodopentane (0.63 mL, 4.21 mmol, 5 eq) and potassium carbonate (116mg, 0.84 mmol, 1 eq) were added to a solution of phenol 54 (400 mg, 0.84mmol) in acetone (4 mL, dried over molecular sieves). The reactionmixture was then warmed to 60° C. and stirred for 6 hours. Acetone wasremoved by rotary evaporation under reduced pressure. The resultingresidue was subjected to flash column chromatography (silica gel; 50/50,v/v, hexane/ethyl acetate). Pure fractions were collected and combinedand excess eluent was removed to provide 55 in 90% yield. LC/MS, 3.90min (ES+) m/z (relative intensity) 670.91 ([M]⁺, 100). ¹H NMR (400 MHz,CDCl₃) δ 7.23 (s, 1H), 6.69 (s, 1H), 6.60 (s, 1H), 5.87 (d, J=8.8 Hz,1H), 5.83-5.68 (m, J=5.6 Hz, 1H), 5.15-5.01 (m, 2H), 4.67-4.58 (m, 1H),4.45-4.35 (m, 1H), 4.04-3.93 (m, 2H), 3.91 (s, 3H), 3.73 (td, J=10.0,3.8 Hz, 1H), 3.25-3.14 (m, J=8.5, 7.0 Hz, 2H), 2.92 (dd, J=16.8, 10.3Hz, 1H), 2.38 (d, J=16.8 Hz, 1H), 1.95-1.81 (m, 4H), 1.77 (s, 3H),1.64-1.49 (m, 2H), 0.88 (s, 9H), 0.25 (s, 3H), 0.23 (s, 3H).

(iii)(11S,11aS)-4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(70)

(a) Allyl3-(2-(2-(4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)hydrazinyl)propanamido)-4-methyl-2-oxopentanoate(56)

Triethylamine (2.23 mL, 18.04 mmol, 2.2 eq) was added to a stirredsolution of the amine 49 (4 g, 8.20 mmol) and triphosgene (778 mg, 2.95mmol, 0.36 eq) in dry tetrahydrofuran (40 mL) at 5° C. (ice bath). Theprogress of the isocyanate reaction was monitored by periodicallyremoving aliquots from the reaction mixture and quenching with methanoland performing LC/MS analysis. Once the isocyanate formation wascomplete a solution of the alloc-Val-Ala-PABOH (4.12 g, 12.30 mmol, 1.5eq) and triethylamine (1.52 mL, 12.30 mmol, 1.5 eq) in drytetrahydrofuran (40 mL) was rapidly added by injection to the freshlyprepared isocyanate. The reaction mixture was allowed to stir at 40° C.for 4 hours. Excess solvent was removed by rotary evaporation underreduced pressure. The resulting residue was subjected to flash columnchromatography (silica gel; gradient, 1% methanol to 5% methanol indichloromethane). (Alternative chromatography conditions using EtOAc andHexane have also been successful). Pure fractions were collected andcombined and excess eluent was removed by rotary evaporation underreduced pressure to give the product 56 (3.9 g, 50%). LC/MS, 4.23 min(ES+) m/z (relative intensity) 952.36 ([M+H]⁺., 100); ¹H NMR (400 MHz,CDCl₃) δ 8.62 (br s, 1H), 8.46 (s, 1H), 7.77 (br s, 1H), 7.53 (d, J=8.4Hz, 2H), 7.32 (d, J=8.5 Hz, 2H), 6.76 (s, 1H), 6.57 (d, J=7.6 Hz, 1H),6.17 (s, 1H), 6.03-5.83 (m, 1H), 5.26 (dd, J=33.8, 13.5 Hz, 3H), 5.10(s, 2H), 4.70-4.60 (m, 2H), 4.58 (dd, J=5.7, 1.3 Hz, 2H), 4.06-3.99 (m,1H), 3.92 (s, 1H), 3.82-3.71 (m, 1H), 3.75 (s, 3H), 2.79-2.64 (m, 1H),2.54 (d, J=12.9 Hz, 1H), 2.16 (dq, J=13.5, 6.7 Hz, 1H), 1.67 (s, 3H),1.46 (d, J=7.0 Hz, 3H), 1.35-1.24 (m, 3H), 1.12 (s, 9H), 1.10 (s, 9H),0.97 (d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H), 0.87 (s, 9H), 0.07-−0.02(m, 6H).

(b) Allyl3-(2-(2-(4-((((2-((S)-2-(hydroxymethyl)-4-methyl-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)hydrazinyl)propanamido)-4-methyl-2-oxopentanoate(57)

The TBS ether 56 (1.32 g, 1.38 mmol) was dissolved in a 7:1:1:2 mixtureof acetic acid/methanol/tetrahydrofuran/water (14:2:2:4 mL) and allowedto stir at room temperature. After 3 hours no more starting material wasobserved by LC/MS. The reaction mixture was diluted with ethyl acetate(25 mL) and washed sequentially with water, saturated aqueous sodiumbicarbonate and brine. The organic phase was dried over magnesiumsulphate filtered and excess ethyl acetate removed by rotary evaporationunder reduced pressure. The resulting residue was subjected to flashcolumn chromatography (silica gel, 2% methanol in dichloromethane). Purefractions were collected and combined and excess eluent was removed byrotary evaporation under reduced pressure to afford the desired product57 (920 mg, 80%). LC/MS, 3.60 min (ES+) m/z (relative intensity) 838.18([M+H]⁺., 100). ¹H NMR (400 MHz, CDCl₃) δ 8.55 (s, 1H), 8.35 (s, 1H),7.68 (s, 1H), 7.52 (d, J=8.1 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H), 6.77 (s,1H), 6.71 (d, J=7.5 Hz, 1H), 6.13 (s, 1H), 5.97-5.82 (m, J=5.7 Hz, 1H),5.41-5.15 (m, 3H), 5.10 (d, J=3.5 Hz, 2H), 4.76-4.42 (m, 5H), 4.03 (t,J=6.6 Hz, 1H), 3.77 (s, 5H), 2.84 (dd, J=16.7, 10.4 Hz, 1H), 2.26-2.08(m, 2H), 1.68 (s, 3H), 1.44 (d, J=7.0 Hz, 3H), 1.30 (dt, J=14.7, 7.4 Hz,3H), 1.12 (s, 9H), 1.10 (s, 9H), 0.96 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8Hz, 3H).

(c)(11S,11aS)-4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-hydroxy-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(58)

Dimethyl sulphoxide (0.2 mL, 2.75 mmol, 2.5 eq) was added dropwise to asolution of oxalyl chloride (0.11 mL, 1.32 mmol, 1.2 eq) in drydichloromethane (7 mL) at −78° C. (dry ice/acetone bath) under anatmosphere of argon. After 10 minutes a solution of 57 (920 mg, 1.10mmol) in dry dichloromethane (5 mL) was added slowly with thetemperature still at −78° C. After 15 min triethylamine (0.77 mL, driedover 4 Å molecular sieves, 5.50 mmol, 5 eq) was added dropwise and thedry ice/acetone bath was removed. The reaction mixture was allowed toreach room temperature and was extracted with cold hydrochloric acid(0.1 M), saturated aqueous sodium bicarbonate and brine. The organicphase was dried over magnesium sulphate, filtered and excessdichloromethane was removed by rotary evaporation under reducedpressure. The resulting residue was subjected to column flashchromatography (silica gel; gradient 2% methanol to 5% methanol indichloromethane). Pure fractions were collected and combined and removalof excess eluent by rotary evaporation under reduced pressure affordedthe product 58 (550 mg, 60%). LC/MS, 3.43 min (ES+) m/z (relativeintensity) 836.01 ([M]⁺., 100). ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H),7.52-7.40 (m, 2H), 7.21-7.08 (m, J=11.5 Hz, 2H), 6.67 (s, 1H), 6.60-6.47(m, J=7.4 Hz, 1H), 5.97-5.83 (m, 1H), 5.79-5.66 (m, 1H), 5.38-4.90 (m,6H), 4.68-4.52 (m, J=18.4, 5.5 Hz, 4H), 4.04-3.94 (m, J=6.5 Hz, 1H),3.87-3.76 (m, 5H), 3.00-2.88 (m, 1H), 2.66-2.49 (m, 2H), 2.21-2.08 (m,2H), 1.76 (s, 3H), 1.45 (d, J=7.0 Hz, 3H), 1.09-0.98 (m, J=8.9 Hz, 18H),0.96 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.9 Hz, 3H).

(d)(11S,11aS)-4-(2-(1-((1-(Allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-8-((triisopropylsilyl)oxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(59)

Tert-butyldimethylsilyltriflate (0.38 mL, 1.62 mmol, 3 eq) was added toa solution of compound 58 (450 mg, 0.54 mmol) and 2,6-lutidine (0.25 mL,2.16 mmol, 4 eq) in dry dichloromethane (5 mL) at 0° C. under argon.After 10 min, the cold bath was removed and the reaction mixture wasstirred at room temperature for 1 hour. The reaction mixture wasextracted with water, saturated aqueous sodium bicarbonate and brine.The organic phase was dried over magnesium sulphate, filtered and excesssolvent was removed by rotary evaporation under reduced pressure. Theresulting residue was subjected to column flash chromatography (silicagel; 50/50 v/v hexane/ethyl acetate). Pure fractions were collected andcombined and excess eluent was removed by rotary evaporation underreduced pressure to give the product 59 (334 mg, 65%). LC/MS, 4.18 min(ES+) m/z (relative intensity) 950.50 ([M]⁺, 100). ¹H NMR (400 MHz,CDCl₃) δ 8.53 (s, 1H), 8.02 (s, 1H), 7.44 (d, J=7.6 Hz, 2H), 7.21 (s,1H), 7.08 (d, J=8.2 Hz, 2H), 6.72-6.61 (m, J=8.9 Hz, 2H), 6.16 (s, 1H),5.97-5.79 (m, J=24.4, 7.5 Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5Hz, 1H), 4.69-4.60 (m, 1H), 4.57 (s, 1H), 4.03 (t, J=6.7 Hz, 1H), 3.87(s, 3H), 3.74 (td, J=9.6, 3.6 Hz, 1H), 2.43-2.09 (m, J=34.8, 19.4, 11.7Hz, 3H), 1.76 (s, 3H), 1.43 (d, J=6.9 Hz, 3H), 1.30-1.21 (m, 3H), 0.97(d, J=6.7 Hz, 3H), 0.92 (t, J=8.4 Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H),0.12 (s, 3H).

(e)(11S,11aS)-4-(2-(1-((1-(Allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(60)

Lithium acetate (50 mg, 0.49 mmol) was added to a solution of compound59 (470 mg, 0.49 mmol) in wet dimethylformamide (4 mL, 50:1 DMF/water).After 4 hours, the reaction was complete and the reaction mixture wasdiluted with ethyl acetate and washed with citric acid (pH˜3), water andbrine. The organic layer was dried over magnesium sulphate filtered andexcess ethyl acetate was removed by rotary evaporation under reducedpressure. The resulting residue was subjected to column flashchromatography (silica gel; gradient, 50/50 to 25/75 v/v hexane/ethylacetate). Pure fractions were collected and combined and excess eluentwas removed by rotary evaporation under reduced pressure to give theproduct 60 (400 mg, quantitative). LC/MS, 3.32 min (ES+) m/z (relativeintensity) 794.18 ([M+H]⁺., 100). 25 ¹H NMR (400 MHz, CDCl₃) δ 8.53 (s,1H), 8.02 (s, 1H), 7.44 (d, J=7.6 Hz, 2H), 7.21 (s, 1H), 7.08 (d, J=8.2Hz, 2H), 6.72-6.61 (m, J=8.9 Hz, 2H), 6.16 (s, 1H), 5.97-5.79 (m,J=24.4, 7.5 Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5 Hz, 1H),4.69-4.60 (m, 1H), 4.57 (s, 1H), 4.03 (t, J=6.7 Hz, 1H), 3.87 (s, 3H),3.74 (td, J=9.6, 3.6 Hz, 1H), 2.43-2.09 (m, J=34.8, 19.4, 11.7 Hz, 3H),1.76 (s, 3H), 1.43 (d, J=6.9 Hz, 3H), 1.30-1.21 (m, 3H), 0.97 (d, J=6.7Hz, 3H), 0.92 (t, J=8.4 Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H), 0.12 (s,3H).

(iv)(11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(64)

(a) (11S)-ally8-((5-(((11S)-10-(((4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl)oxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(61)

Potassium carbonate (70 mg, 0.504 mmol, 1 eq) was added to a solution of55 (370 mg, 0.552 mmol, 1.2 eq) and phenol 60 (400 mg, 0.504 mmol) indry acetone (25 mL). The reaction was stirred 8 hours at 70° C. TheLC/MS showed that all the starting material was not consumed, so thereaction was allowed to stir overnight at room temperature and stirredfor an additional 2 hours the next day. Acetone was removed by rotaryevaporation under reduced pressure. The resulting residue was subjectedto flash column chromatography (silica gel; 80% ethyl acetate in hexaneto 100% ethyl acetate). Pure fractions were collected and combined andexcess eluent was removed by rotary evaporation under reduced pressureto give the product 61 (385 mg, 57%). LC/MS, 4.07 min (ES+) m/z(relative intensity) 1336.55 ([M+H]⁺., 50).

(b) (11S)-allyl8-((5-(((11S)-10-(((4-(2-(1-((1-(allyloxy)-4-methyl-1,2-dioxopentan-3-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(62)

Tetra-n-butylammonium fluoride (1 M, 0.34 mL, 0.34 mmol, 2 eq) was addedto a solution of 61 (230 mg, 0.172 mmol) in dry tetrahydrofuran (3 mL).The starting material was totally consumed after 10 minutes. Thereaction mixture was diluted with ethyl acetate (30 mL) and washedsequentially with water and brine. The organic phase was dried overmagnesium sulphate filtered and excess ethyl acetate removed by rotaryevaporation under reduced pressure. The resulting residue 62 was used asa crude mixture for the next reaction. LC/MS, 2.87 min (ES+) m/z(relative intensity) 1108.11 ([M+H]⁺., 100).

(c)(11S)-4-(2-(1-((1-amino-3-methyl-1-oxobutan-2-yl)amino)-1-oxopropan-2-yl)hydrazinyl)benzyl11-hydroxy-7-methoxy-8-((5-((7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(63)

Tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol, 0.06 eq) wasadded to a solution of crude 62 (0.172 mmol) and pyrrolidine (36 μL,0.43 mmol, 2.5 eq) in dry dichloromethane (10 mL). The reaction mixturewas stirred 20 minutes and diluted with dichloromethane and washedsequentially with saturated aqueous ammonium chloride and brine. Theorganic phase was dried over magnesium sulphate filtered and excessdichloromethane removed by rotary evaporation under reduced pressure.The resulting residue 63 was used as a crude mixture for the nextreaction. LC/MS, 2.38 min (ES+) m/z (relative intensity) 922.16([M+H]⁺., 40).

(d)(11S,11aS)-4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(64)

1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCl, 33 mg, 0.172 mmol)was added to a solution of crude 63 (0.172 mmol) and Mal-(PEG)₈-acid(100 mg, 0.172 mmol) in dry dichloromethane (10 mL). The reaction wasstirred for 2 hours and the presence of starting material was no longerobserved by LC/MS. The reaction was diluted with dichloromethane andwashed sequentially with water and brine. The organic phase was driedover magnesium sulphate filtered and excess dichloromethane removed byrotary evaporation under reduced pressure. The resulting residue wassubjected to flash column chromatography (silica gel; 100% chloroform to10% methanol in chloroform). Pure fractions were collected and combinedand excess eluent was removed by rotary evaporation under reducedpressure to give 64 (E) (60 mg, 25% over 3 steps).

Example 8

Compound 65 is compound 79 of WO 2011/130598

(11S)-4-(1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl11-hydroxy-7-methoxy-8-(3-((7-methoxy-5-oxo-2-((E)-prop-1-en-1-yl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2-((E)-prop-1-en-1-yl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(66)

N,N′-diisopropylcarbodiimide (DIC, 4.71 μL, 0.0304 mmol) was added to asolution of amine 65 (0.0276 mmol) and Iodo-(PEG)₄-acid (13.1 mg, 0.0304mmol) in dry dichloromethane (0.8 mL). The reaction was stirred for 3hours and the presence of starting material was no longer observed byLC/MS. The reaction mixture was directly loaded onto a thin-layerchromatography (TLC) plate and purified by prep-TLC (10% methanol inchloroform). Pure bands were scraped off the TLC plate, taken up in 10%methanol in chloroform, filtered and excess eluent removed by rotaryevaporation under reduced pressure to give 66 (D) (20.9 mg, 56%). LC/MS,method 2, 3.08 min (ES+) m/z (relative intensity) 1361.16 ([M+H]⁺, 100).

General Experimental Methods for Example 9

LCMS data were obtained using an Agilent 1200 series LC/MS with anAgilent 6110 quadrupole MS, with Electrospray ionisation. Mobile phaseA—0.1% Acetic acid in water. Mobile Phase B—0.1% in acetonitrile. Flowrate of 1.00 ml/min. Gradient from 5% B rising up to 95% B over 3minutes, remaining at 95% B for 1 minute and then back down to 5% B over6 seconds. The total run time is 5 minutes. Column: Phenomenex Gemini-NX3 μm C18, 30×2.00 mm. Chromatograms based on UV detection at 254 nm.Mass Spectra were achieved using the MS in positive mode. Proton NMRchemical shift values were measured on the delta scale at 400 MHz usinga Bruker AV400. The following abbreviations have been used: s, singlet;d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Couplingconstants are reported in Hz. Unless otherwise stated, columnchromatography (by the flash procedure) were performed on MerckKieselgel silica (Art. 9385). Mass spectroscopy (MS) data were collectedusing a Waters Micromass LCT instrument coupled to a Waters 2795 HPLCseparations module. Thin Layer Chromatography (TLC) was performed onsilica gel aluminium plates (Merck 60, F₂₅₄). All other chemicals andsolvents were purchased from Sigma-Aldrich or Fisher Scientific and wereused as supplied without further purification.

Optical rotations were measured on an ADP 220 polarimeter (BellinghamStanley Ltd.) and concentrations (c) are given in g/100 mL. Meltingpoints were measured using a digital melting point apparatus(Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 Kusing a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively.Chemical shifts are reported relative to TMS (δ=0.0 ppm), and signalsare designated as s (singlet), d (doublet), t (triplet), dt (doubletriplet), dd (doublet of doublets), ddd (double doublet of doublets) orm (multiplet), with coupling constants given in Hertz (Hz). Massspectroscopy (MS) data were collected using a Waters Micromass ZQinstrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. WatersMicromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35;Extractor (V), 3.0; Source temperature (° C.), 100; DesolvationTemperature (° C.), 200; Cone flow rate (L/h), 50; De-solvation flowrate (L/h), 250. High-resolution mass spectroscopy (HRMS) data wererecorded on a Waters Micromass QTOF Global in positive W-mode usingmetal-coated borosilicate glass tips to introduce the samples into theinstrument. Thin Layer Chromatography (TLC) was performed on silica gelaluminium plates (Merck 60, F₂₅₄), and flash chromatography utilisedsilica gel (Merck 60, 230-400 mesh ASTM). Except for the HOBt(NovaBiochem) and solid-supported reagents (Argonaut), all otherchemicals and solvents were purchased from Sigma-Aldrich and were usedas supplied without further purification. Anhydrous solvents wereprepared by distillation under a dry nitrogen atmosphere in the presenceof an appropriate drying agent, and were stored over 4 Å molecularsieves or sodium wire. Petroleum ether refers to the fraction boiling at40-60° C.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run usinga mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B)(formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then5% B to 95% B within 3 min. The composition was held for 0.5 min at 95%B, and then returned to 5% B in 0.3 minutes. Total gradient run timeequals 5 min. Flow rate 3.0 mL/min, 400 μL was split via a zero deadvolume tee piece which passes into the mass spectrometer. Wavelengthdetection range: 220 to 400 nm. Function type: diode array (535 scans).Column: Phenomenex® Onyx Monolithic C18 50×4.60 mm

Example 9 (i) Key Intermediates

(a-i) (S)-2-(allyloxycarbonylamino)-3-methylbutanoic acid (12)

Allyl chloroformate (36.2 ml, 340.59 mmol, 1.2 eq) was added dropwise toa stirred solution of L-valine (I1)(33.25 g, 283.82 mmol, 1.0 eq) andpotassium carbonate (59.27 g, 425.74 mmol, 1.5 eq) in water (650 mL) andTHF (650 mL). The reaction mixture was stirred at room temperature for18 hours, then the solvent was concentrated under reduced pressure andthe remaining solution extracted with diethyl ether (3×100 mL). Theaqueous portion was acidified to pH 2 with conc. HCl and extracted withDCM (3×100 mL). The combined organics were washed with brine, dried overMgSO₄, filtered and concentrated under reduced pressure to afford theproduct as a colourless oil (57.1 g, assumed 100% yield). LC/MS (1.966min (ES⁺)), m/z: 202.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.57 (br s,1H), 7.43 (d, 1H, J=8.6 Hz), 5.96-5.86 (m, 1H), 5.30 (ddd, 1H, J=17.2,3.4, 1.7 Hz), 5.18 (ddd, 1H, J=10.4, 2.9, 1.6 Hz), 4.48 (dt, 2H, J=5.3,1.5 Hz), 3.85 (dd, 1H, J=8.6, 6.0 Hz), 2.03 (oct, 1H, J=6.6 Hz), 0.89(d, 3H, J=6.4 Hz), 0.87 (d, 3H, J=6.5 Hz).

(a-ii) (S)-2,5-dioxopyrrolidin-1-yl2-(allyloxycarbonylamino)-3-methylbutanoate (13)

To a stirred solution of the protected acid 12 (60.6 g, 301.16 mmol, 1.0eq) and N-hydroxysuccinimide (34.66 g, 301.16 mmol, 1.0 eq) in dry THF(800 mL) was added dicyclohexylcarbodiimide (62.14 g, 301.16 mmol, 1eq). The reaction was stirred for 18 hours at room temperature. Thereaction mixture was then filtered, the solid washed with THF and thecombined filtrate was concentrated under reduced pressure. The residuewas re-dissolved in DCM and left to stand at 0° C. for 30 minutes. Thesuspension was filtered and washed with cold DCM. Concentration of thefiltrate under reduced pressure afforded the product as a viscouscolourless oil (84.7 g, assumed 100% yield) which was used in the nextstep without further purification. LC/MS (2.194 min (ES⁺)), m/z: 321.0[M+Na]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.0 (d, 1H, J=8.3 Hz), 5.97-5.87(m, 1H), 5.30 (ddd, 1H, J=17.2, 3.0, 1.7 Hz), 5.19 (ddd, 1H, J=10.4,2.7, 1.4 Hz), 4.52 (dt, 2H, J=5.3, 1.4 Hz), 4.32 (dd, 1H, J=8.3, 6.6Hz), 2.81 (m, 4H), 2.18 (oct, 1H, J=6.7 Hz), 1.00 (d, 6H, J=6.8 Hz),

(a-iii)(S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanoic acid(14)

A solution of succinimide ester 13(12.99 g, 43.55 mmol, 1.0 eq) in THF(50 mL) was added to a solution of L-alanine (4.07 g, 45.73 mmol, 1.05eq) and NaHCO₃ (4.02 g, 47.90 mmol, 1.1 eq) in THF (100 mL) and H₂O (100mL). The mixture was stirred at room temperature for 72 hours when theTHF was removed under reduced pressure. The pH was adjusted to 3-4 withcitric acid to precipitate a white gum. After extraction with ethylacetate (6×150 mL), the combined organics were washed with H₂O (200 mL),dried over MgSO₄, filtered and concentrated under reduced pressure.Trituration with diethyl ether afforded the product as a white powderwhich was collected by filtration and washed with diethyl ether (5.78 g,49%). LC/MS (1.925 min (ES⁺)), m/z: 273.1 [M+H]⁺. ¹H NMR (400 MHz,DMSO-d₆) δ 12.47 (br s, 1H), 8.17 (d, 1H, J=6.8 Hz), 7.16 (d, 1H, J=9.0Hz), 5.95-5.85 (m, 1H), 5.29 (dd, 1H, J=17.2, 1.7 Hz), 5.17 (dd, 1H,J=10.4, 1.5 Hz), 4.46 (m, 2H), 4.18 (quin, 1H, J=7.2 Hz), 3.87 (dd, 1H,J=9.0, 7.1 Hz), 1.95 (oct, 1H, J=6.8 Hz), 1.26 (d, 3H, J=7.3 Hz), 0.88(d, 3H, J=6.8 Hz), 0.83 (d, 3H, J=6.8 Hz).

(a-iv)Allyl(S)-1-((S)-1-(4-(hydroxymethyl)phenylamino)-1-oxopropan-2-ylamino)-3-methyl-1-oxobutan-2-ylcarbamate(15)

EEDQ (5.51 g, 22.29 mmol, 1.05 eq) was added to a solution ofp-aminobenzyl alcohol (2.74 g, 22.29 mmol, 1.05 eq) and acid 14 (5.78 g,21.23 mmol, 1 eq) in dry THF (100 mL). and stirred at room temperaturefor 72 hours. The reaction mixture was then concentrated under reducedpressure and the resulting brown solid was triturated with diethyl etherand filtered with subsequent washing with an excess of diethyl ether toafford the product as an off-white solid (7.1 g, 88%). LC/MS (1.980 min(ES⁺)), m/z: 378.0 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.89 (br s, 1H),8.13 (d, 1H, J=7.0 Hz), 7.52 (d, 2H, J=8.5 Hz), 7.26 (m, 1H), 7.23 (d,2H, J=8.5 Hz), 5.91 (m, 1H), 5.30 (m, 1H), 5.17 (m, 1H), 4.46 (m, 2H),5.09 (t, 1H, J=5.6 Hz), 4.48 (m, 2H), 4.42 (m, 3H), 3.89 (dd, 1H, J=8.6,6.8 Hz), 1.97 (m, 1H), 1.30 (d, 3H, J=7.1 Hz), 0.88 (d, 3H, J=6.8 Hz),0.83 (d, 3H, J=6.7 Hz).

1-iodo-2-oxo-6,9,12,15-tetraoxa-3-azaoctadecan-18-oic acid (17)

A solution of iodoacetic anhydride (0.250 g, 0.706 mmol, 1.1 eq) in dryDCM (1 mL) was added to amino-PEG₍₄₎-acid 16 (0.170 g, 0.642 mmol, 1.0eq) in DCM (1 mL). The mixture was stirred in the dark at roomtemperature overnight. The reaction mixture was washed with 0.1 M HCl,water, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography (silica gel,3% MeOH and 0.1% formic acid in chloroform to 10% MeOH and 0.1% formicacid in chloroform) to afford the product as an orange oil (0.118 g,42%). LC/MS (1.623 min (ES⁺)), m/z: 433.98 [M+H]⁺. ¹H NMR (400 MHz,CDCl₃) δ 8.069 (s, 1H), 7.22 (br s, 1H), 3.79 (t, 2H, J=5.8 Hz), 3.74(s, 2H), 3.72-3.58 (m, 14H), 3.50-3.46 (m, 2H), 2.62 (t, 2H, J=5.8 Hz).

(ii) (11S,11aS)-allyl11-(tert-butyldimethylsilyloxy)-8-(3-iodopropoxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(74)

(a)(S)-5-((tert-butyldimethylsilyloxy)methyl)-1-(5-methoxy-2-nitro-4-(triisopropylsilyloxy)benzoyl)-4,5-dihydro-1H-pyrrol-3-yltrifluoromethanesulfonate (47)

Triflic anhydride (28.4 g, 100.0 mmol, 3.0 eq) was added dropwise, over25 mins, to a vigorously stirred solution of the ketone 46 (19.5 g, 30.0mmol, 1.0 eq) in DCM (550 mL) containing 2,6-lutidine (14.4 g, 130.0mmol, 4.0 eq) at −50° C. The reaction mixture was stirred for 1.5 hourswhen LC/MS indicated complete reaction. The organic phase was washedsuccessively with water (100 mL), saturated sodium bicarbonate (150 mL),brine (50 mL), and the organic phase was dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography (silica gel, 90/10 v/v n-hexane/EtOAc) to afford theproduct as a pale yellow oil (19.5 g, 82%). LC/MS (4.391 min (ES⁺)),m/z: 713.25 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.68 (s, 1H), 6.72 (s,1H), 6.02 (t, 1H, J=1.9 Hz), 4.75 (m, 1H), 4.05 (m, 2H), 3.87 (s, 3H),3.15 (ddd, 1H, J=16.2, 10.3, 2.3 Hz), 2.96 (ddd, 1H, J=16.2, 4.0, 1.6Hz), 1.28-1.21 (m, 3H), 1.07 (d, 18H, J=7.2 Hz), 0.88 (s, 9H), 0.09 (s,3H), 0.08 (s, 3H).

(b)(S,E)-(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrol-1-yl)(5-methoxy-2-nitro-4-(triisopropylsilyloxy)phenyl)methanone(67)

Tetrakis(triphenylphosphine)palladium(0) (0.41 g, 0.35 mmol, 0.03 eq)was added to a mixture of the triflate 47 (8.4 g, 11.8 mmol, 1.0 eq),E-1-propene-1-ylboronic acid (1.42 g, 16.5 mmol, 1.4 eq) and potassiumphosphate (5.0 g, 23.6 mmol, 2.0 eq) in dry dioxane (60 mL) under anitrogen atmosphere. The mixture was stirred at 25° C. for 120 mins whenLC/MS indicated complete reaction. Ethyl acetate (120 mL) and water (120mL) were added, the organic phase was removed, washed with brine (20mL), dried over MgSO₄, filtered and concentrated under reduced pressure.The residue was purified by flash chromatography (silica gel, 95/5 v/vn-hexane/EtOAc to 90/10 v/v n-hexane/EtOAc) to afford the product as ayellow foam (4.96 g, 70%). LC/MS (4.477 min (ES⁺)), m/z: 605.0 [M+H]⁺.¹H NMR (400 MHz, CDCl₃) δ 7.67 (s, 1H), 6.74 (s, 1H), 5.93 (d, 1H,J=15.4 Hz), 5.67 (s, 1H), 4.65 (m, 1H), 4.04 (m, 2H), 3.86 (s, 3H), 2.85(m, 1H), 2.71 (m, 1H), 1.72 (dd, 3H, J=6.8, 1.0 Hz), 1.30-1.22 (m, 3H),1.07 (d, 18H, J=7.2 Hz), 0.87 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H).

(c) (S,E)-(2-amino-5-methoxy-4-(triisopropylsilyloxy)phenyl)(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrol-1-yl)methanone(68)

Zinc dust (22.0 g, 0.33 mol, 37 eq) was added, in portions over 20 mins,to a solution of the propenyl intermediate 67 (5.5 g, 9.1 mmol, 1.0 eq)in 5% v/v formic acid/ethanol (55 mL), using an ice bath to maintain thetemperature between 25-30° C. After 30 mins, the reaction mixture wasfiltered through a short bed of Celite®. The Celite® was washed withethyl acetate (65 mL) and the combined organics were washed successivelywith water (35 mL), saturated sodium bicarbonate (35 mL) and brine (10mL). The organic phase was dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was purified by flash chromatography(silica gel, 90/10 v/v n-hexane/EtOAc) to afford the product as a paleyellow oil (3.6 g, 69.0%). LC/MS (4.439 min (ES⁺)), m/z: 575.2 [M+H]⁺.¹H NMR (400 MHz, CDCl₃) δ 6.75 (m, 1H), 6.40 (br s, 1H), 6.28 (m, 1H),6.11 (d, 1H, J=15.4 Hz), 5.53 (m, 1H), 4.67 (m, 1H), 4.36 (m, 2H), 3.93(br s, 1H), 3.84 (br s, 1H), 3.73 (s, 3H), 2.86 (dd, 1H, J=15.7, 10.4Hz), 2.73 (dd, 1H, J=15.9, 4.5 Hz), 1.80 (dd, 3H, J=6.8, 1.3 Hz),1.35-1.23 (m, 3H), 1.12 (d, 18H, J=7.3 Hz), 0.89 (s, 9H), 0.08 (s, 3H),0.07 (s, 3H).

(d) (S,E)-allyl2-(2-((tert-butyldimethylsilyloxy)methyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-(triisopropylsilyloxy)phenylcarbamate(69)

Allyl chloroformate (0.83 g, 6.88 mmol, 1.1 eq) was added to a solutionof the amine 68 (3.6 g, 6.26 mmol, 1.0 eq) in dry DCM (80 mL) containingdry pyridine (1.09 g, 13.77 mmol, 2.2 eq) at −78° C. The dry ice wasremoved and the reaction mixture allowed to warm to room temperature.After stirring for a further 15 minutes, LC/MS indicated completereaction. The organic phase was washed successively with 0.01N HCl (50mL), saturated sodium bicarbonate (50 mL), brine (10 mL), dried overMgSO₄, filtered and concentrated under reduced pressure to leave a paleyellow oil which was used in the next step without further purification(4.12 g, assumed 100% yield). LC/MS (4.862 min (ES⁺)), m/z: 659.2[M+H]⁺.

(e) (S,E)-allyl2-(2-(hydroxymethyl)-4-(prop-1-enyl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-(triisopropylsilyloxy)phenylcarbamate(70)

The crude intermediate 69 (assumed 100% yield, 4.12 g, 6.25 mmol, 1.0eq) was dissolved in a mixture of acetic acid (70 mL), methanol (10 mL),THF (10 mL) and water (20 mL) and allowed to stir at room temperature.After 6 hours the reaction mixture was diluted with ethyl acetate (500mL) and washed successively with water (2×500 mL), saturated sodiumbicarbonate (300 mL) and brine (50 mL). The organic phase was dried overMgSO₄, filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 1/99 v/v methanol/DCM to5/95 v/v methanol/DCM) to afford the product as a yellow oil and afurther 1 g of unreacted starting material was recovered. This materialwas subjected to the same reaction conditions as above, but was leftstirring for 16 h. After work up and purification, additional productwas isolated (2.7 g, 79%, 2 steps) LC/MS (3.742 min (ES⁺)), m/z: 545.2[M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.38 (m, 1H), 7.72 (m, 1H), 6.81 (s,1H), 6.37 (m, 1H), 6.10 (d, 1H, J=15.8 Hz), 5.97 (m, 1H), 5.53 (m, 1H),5.36 (ddd, 1H, J=17.2, 3.1, 1.5 Hz), 5.25 (ddd, 1H, J=10.4, 2.5, 1.3Hz), 4.78 (m, 1H), 4.65 (dt, 2H, J=5.7, 1.3 Hz), 3.84 (m, 3H), 3.79 (s,3H), 3.04 (dd, 1H, J=16.7, 10.5 Hz), 2.40 (dd, 1H, J=16.0, 4.5 Hz), 1.82(dd, 3H, J=6.8, 1.0 Hz), 1.36-1.26 (m, 3H), 1.14 (d, 18H, J=7.3 Hz).

(f) (11S,11aS)-allyl11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(71)

Dry dimethyl sulfoxide (1.16 g, 14.87 mmol, 3.0 eq) was added dropwiseto a solution of oxalyl chloride (0.94 g, 7.43 mmol, 1.5 eq) in DCM (25mL) at −78° C. under an atmosphere of nitrogen. Maintaining thetemperature at −78° C., after 10 mins a solution of the primary alcohol70 (2.7 g, 4.96 mmol, 1.0 eq) in DCM (20 mL) was added dropwise. After afurther 15 mins, dry triethylamine (2.5 g, 24.78 mmol, 5.0 eq) wasadded, and the reaction mixture allowed to warm to room temperature. Thereaction mixture was washed successively with cold 0.1N HCl (50 mL),saturated sodium hydrogen carbonate (50 mL) and brine (10 mL) and theorganic layer was dried over MgSO₄, filtered and concentrated underreduced pressure to afford the product as a yellow oil which was used inthe next step without further purification (2.68 g, assumed 100% yield).LC/MS (3.548 min (ES⁺)), m/z: 543.2 [M+H]⁺.

(g) (11S,11aS)-allyl11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(72)

Tert-butyldimethylsilyltrifluoromethane sulfonate (3.93 g, 14.87 mmol,3.0 eq) was added to a solution of the carbinolamine 71 (assumed 100%yield, 2.68 g, 4.96 mmol, 1.0 eq) and 2,6-lutidine (2.12 g, 19.83 mmol,4.0 eq) in dry DCM (40 mL) at 0° C. under an atmosphere of nitrogen.After 10 minutes, the reaction mixture was allowed to warm to roomtemperature and stirred for a further 60 minutes. The organic phase waswashed successively with water (10 mL), saturated sodium bicarbonate (10mL) and brine (5 mL), dried over MgSO₄, filtered and concentrated underreduced pressure. The residue was purified by flash chromatography(silica gel, chloroform to 2/98 v/v Methanol/chloroform) to afford theproduct as a yellow oil (2.0 g, 63%, 2 steps). LC/MS (4.748 min (ES⁺)),m/z: 657.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.19 (s, 1H), 6.86 (m, 1H),6.66 (s, 1H), 6.22 (d, 1H, J=15.4 Hz), 5.81 (d, 1H, J=8.8 Hz), 5.78 (m,1H), 5.48 (m, 1H), 5.11 (d, 1H, J=5.0 Hz), 5.08 (m, 1H), 4.58 (dd, 1H,J=13.4, 5.4 Hz), 4.35 (dd, 1H, J=13.2, 5.7 Hz), 3.83 (s, 3H), 3.76 (s,1H), 3.00 (dd, 1H, J=15.6, 11.0 Hz), 2.53 (m, 1H), 1.81 (dd, 3H, J=6.8,0.9 Hz), 1.30-1.18 (m, 3H), 1.08 (d, 9H, J=2.3 Hz), 1.06 (d, 9H, J=2.3Hz), 0.86 (s, 9H), 0.25 (s, 3H), 0.18 (s, 3H).

(h) (11S,11aS)-allyl11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(73)

Lithium acetate dihydrate (0.31 g, 3.04 mmol, 1.0 eq) was added to asolution of the diazepine 72 (2.0 g, 3.04 mmol, 1.0 eq) in wet DMF (20mL) at 25° C. and stirred for 4 hours. The reaction mixture was dilutedwith ethyl acetate (200 mL) and washed successively with 0.1M citricacid (50 mL, pH 3), water (50 mL) and brine (10 mL), dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 50/50 v/v n-hexane/EtOActo 25/75 v/v n-hexane/EtOAc) to afford the product as a pale yellowsolid (0.68 g, 45%). LC/MS (3.352 min (ES⁺)), m/z: 501.1 [M+H]⁺. ¹H NMR(400 MHz, CDCl₃) δ 7.02 (s, 1H), 6.66 (m, 1H), 6.53 (s, 1H), 6.03 (d,1H, J=15.5 Hz), 5.80 (s, 1H), 5.63 (d, 1H, J=8.9 Hz), 5.55 (m, 1H), 5.29(m, 1H), 4.87 (m, 2H), 4.39 (dd, 1H, J=13.5, 4.2 Hz), 4.20 (dd, 1H,J=13.2, 5.7 Hz), 3.73 (s, 3H), 3.59 (m, 1H), 2.81 (dd, 1H, J=16.1, 10.5Hz), 2.35 (d, 1H, J=15.7 Hz), 1.61 (d, 3H, J=6.4 Hz), 0.67 (s, 9H), 0.05(s, 3H), 0.00 (s, 3H).

(i) (11S,11aS)-allyl11-(tert-butyldimethylsilyloxy)-8-(3-iodopropoxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(74)

Diiodopropane (0.295 g, 1.00 mmol, 5.0 eq) and potassium carbonate(0.028 g, 0.20 mmol, 1.0 eq) were added to a solution of the phenol 33(0.100 g, 0.020 mmol, 1.0 eq) in dry acetone (5 mL). The reactionmixture was heated at 60° C. for 6 hours when LC/MS showed completereaction. The reaction mixture was concentrated to dryness under reducedpressure and the residue was purified by flash chromatography (silicagel, 75/25 v/v n-hexane/EtOAc to 50/50 v/v n-hexane/EtOAc) to afford theproduct as a colourless oil (0.074 g, 56%). LC/MS (3.853 min (ES⁺)),m/z: 669.0 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1H), 6.90 (s, 1H),6.68 (s, 1H), 6.24 (d, 1H, J=15.3 Hz), 5.87 (d, 1H, J=8.9 Hz), 5.78 (m,1H), 5.53 (m, 1H), 5.12 (m, 2H), 4.65 (m, 2H), 4.41 (m, 1H), 4.11 (m,1H), 3.93 (s, 3H), 3.81 (m, 1H), 3.40 (t, 2H, J=6.7 Hz), 3.05 (dd, 1H,J=16.3, 10.1 Hz), 2.57 (m, 1H), 2.34 (m, 2H), 1.84 (d, 3H, J=6.6 Hz),0.92 (s, 9H), 0.28 (s, 3H), 0.26 (s, 3H).

(iii) (11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate79)

(a) Allyl((S)-1-(((S)-1-((4-((((2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-((E)-prop-1-en-1-yl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(75)

Triethylamine (0.256 mL, 1.84 mmol, 2.2 eq) was added to a stirredsolution of the amine 68 (0.480 g, 0.835 mmol, 1.0 eq) and triphosgene(0.089 g, 0.301 mmol, 0.36 eq) in dry THF (15 mL) at 5° C. (ice bath).The progress of the isocyanate reaction was monitored by periodicallyremoving aliquots from the reaction mixture and quenching with methanoland performing LCMS analysis. Once the isocyanate reaction was completea solution of Alloc-Val-Ala-PABOH (0.473 g, 1.25 mmol, 1.5 eq) andtriethylamine (0.174 mL, 1.25 mmol, 1.5 eq) in dry THF (10 mL) wasrapidly added by injection to the freshly prepared isocyanate. Thereaction was allowed to stir at 40° C. for 4 hours followed by stirringat room temperature overnight. The mixture was concentrated underreduced pressure, and purified by flash chromatography (silica gel,20/80 v/v n-hexane/EtOAc to 50/50 v/v n-hexane/EtOAc, then 1/99 v/vDCM/MeOH to 5/95 v/v DCM/MeOH) to afford the product as a yellow solid(0.579 g, 71%). LC/MS (4.468 min (ES⁺)), m/z: 978.55 [M+H]⁺. ¹H NMR (400MHz, CDCl₃) δ 8.63 (br s, 1H), 8.42 (s, 1H), 7.78 (br s, 1H), 7.53 (d,2H, J=8.1 Hz), 7.31 (d, 2H, J=8.6 Hz), 6.76 (s, 1H), 6.59 (d, 1H, J=7.6Hz), 6.36 (brs, 1H), 6.04 (d, 1H, J=15.9 Hz), 5.90 (m, 1H), 5.55 (m,1H), 5.33-5.21 (m, 3H), 5.10 (s, 2H), 4.66 (m, 2H), 4.57 (dd, 2H, J=5.6,1.0 Hz), 3.98 (dd, 1H, J=7.3, 6.8 Hz), 3.90 (m, 1H), 3.81 (m, 1H), 3.78(s, 3H), 2.82 (dd, 1H, J=15.4, 9.6 Hz), 2.72 (dd, 1H, J=15.9, 3.5 Hz),2.17 (m, 1H), 1.78 (dd, 3H, J=6.5, 0.8 Hz), 1.46 (d, 3H, J=7.1 Hz), 1.29(m, 3H), 1.11 (d, 18H, J=7.1 Hz), 0.97 (d, 3H, J=6.8 Hz), 0.92 (d, 3H,J=6.8 Hz), 0.83 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H).

(b) Allyl((S)-1-(((S)-1-((4-((((2-((S)-2-(hydroxymethyl)-4-((E)-prop-1-en-1-yl)-2,3-dihydro-1H-pyrrole-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(76)

The silyl ether 75 (1.49 g, 1.52 mmol, 1.0 eq) was dissolved in a7:1:1:2 mixture of acetic acid/methanol/tetrahydrofuran/water (14:2:2:4mL) and allowed to stir at room temperature. After 2 hours the reactionwas diluted with EtOAc (100 mL), washed sequentially with water, aq.sodium bicarbonate then brine. The organic phase was then dried overMgSO₄, filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 100/0 then 99/1 to 92/8v/v DCM/MeOH) to afford the product as an orange solid (1.2 g, 92%).LC/MS (3.649 min (ES⁺)), m/z: 865.44 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ8.44 (s, 1H), 8.35 (s, 1H), 7.69 (br s, 1H), 7.53 (d, 2H, J=8.7 Hz),7.32 (d, 2H, J=8.3 Hz), 6.78 (s, 1H), 6.56 (m, 2H), 6.32 (br s, 1H),6.05 (d, 1H, J=14.9 Hz), 5.90 (m, 1H), 5.56 (m, 1H), 5.30 (m, 2H), 5.22(m, 1H), 5.10 (d, 2H, J=3.1 Hz), 4.73 (m, 1H), 4.64 (m, 1H), 4.57 (d,2H, J=5.8 Hz), 4.01 (m, 1H), 3.79 (m, 2H), 3.76 (s, 3H), 2.98 (dd, 1H,J=16.3, 10.2 Hz), 2.38 (dd, 1H, J=16.6, 4.1 Hz), 2.16 (m, 1H), 1.78 (dd,3H, J=6.8, 0.9 Hz), 1.46 (d, 3H, J=7.1 Hz), 1.29 (m, 3H), 1.11 (d, 18H,J=7.4 Hz), 0.97 (d, 3H, J=6.7 Hz), 0.92 (d, 3H, J=6.8 Hz).

(c)(11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(77)

Dry dimethyl sulfoxide (0.180 g, 2.3 mmol, 3.0 eq) was added dropwise toa solution of oxalyl chloride (0.147 g, 1.1 mmol, 1.5 eq) in DCM (10 mL)at −78° C. under an atmosphere of nitrogen. Maintaining the temperatureat −78° C., after 20 minutes, a solution of the primary alcohol 76(0.666 g, 0.77 mmol, 1.0 eq) in DCM (10 mL) was added dropwise. After afurther 15 minutes, dry triethylamine (0.390 g, 3.85 mmol, 5.0 eq) wasadded, and the reaction mixture allowed to warm to room temperature. Thereaction mixture was washed successively with cold 0.1N HCl (10 mL),saturated sodium hydrogen carbonate (10 mL) and brine (5 mL). Theorganic layer was then dried over MgSO₄, filtered and concentrated underreduced pressure. The residue was then purified by flash chromatography(silica gel, 50/50 v/v n-hexane/EtOAc to 25/75 v/v n-hexane/EtOAc) toafford the product as a white solid (0.356 g, 54%). LC/MS (3.487 min(ES⁺)), m/z: 862.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.34 (br s, 1H),7.47 (d, 2H, J=7.6 Hz), 7.17 (s, 1H), 7.14 (d, 2H, J=7.5 Hz), 6.86 (brs, 1H), 6.65 (br s, 1H), 6.42 (d, 1H, J=7.6 Hz), 6.22 (d, 1H, J=14.4Hz), 5.80 (m, 1H), 5.40 (m, 1H), 5.53 (m, 1H), 5.32 (m, 1H), 5.21 (d,2H, J=9.6 Hz), 5.06 (d, 1H, J=12.3 Hz), 4.90 (m, 1H), 4.58 (m, 3H), 3.98(m, 1H), 3.84 (m, 1H), 3.81 (s, 3H), 3.50 (m, 1H), 3.05 (dd, 1H, J=16.0,10.3 Hz), 2.76 (m, 1H), 2.15 (m, 1H), 1.80 (dd, 3H, J=6.7, 0.8 Hz), 1.44(d, 3H, J=7.1 Hz), 1.16 (m, 3H), 1.01 (d, 18H, J=6.6 Hz), 0.96 (d, 3H,J=6.8 Hz), 0.92 (d, 3H, J=6.8 Hz).

(d)(11S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-8-(triisopropylsilyloxy)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(78)

Tert-butyldimethylsilyltrifluoromethane sulfonate (0.46 g, 1.74 mmol,3.0 eq) was added to a solution of secondary alcohol 77 (0.5 g, 0.58mmol, 1.0 eq) and 2,6-lutidine (0.25 g, 2.32 mmol, 4.0 eq) in dry DCM(10 mL) at 0° C. under an atmosphere of nitrogen. After 10 minutes, thereaction mixture was allowed to warm to room temperature and stirred fora further 120 mins. The organic phase was then washed successively withwater (10 mL), saturated sodium bicarbonate (10 mL) and brine (5 mL),dried over MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography (silica gel, 50/50 v/vn-hexane/EtOAc) to afford the product as a white solid (0.320 g, 57%).LC/MS (4.415 min (ES⁺)), m/z: 976.52 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ8.31 (br s, 1H), 7.48 (d, 2H, J=8.0 Hz), 7.21 (s, 1H), 7.14 (d, 2H,J=8.3 Hz), 6.89 (s, 1H), 6.65 (s, 1H), 6.38 (d, 1H, J=7.3 Hz), 6.25 (d,1H, J=14.6 Hz), 5.93 (m, 1H), 5.85 (d, 1H, J=8.8 Hz), 5.50 (m, 1H), 5.34(m, 1H), 5.24 (m, 2H), 5.15 (d, 1H, J=12.5 Hz), 4.86 (d, 1H, J=12.2 Hz),4.62 (m, 3H), 4.01 (m, 1H), 3.86 (s, 3H), 3.78 (m, 1H), 3.04 (m, 1H),2.56 (m, 1H), 2.20 (m, 1H), 1.84 (dd, 3H, J=6.6, 0.7 Hz), 1.48 (d, 3H,J=6.8 Hz), 1.20 (m, 3H), 1.05 (d, 9H, J=2.9 Hz), 1.03 (d, 9H, J=2.9 Hz),0.99 (d, 3H, J=6.8 Hz), 0.95 (d, 3H, J=6.8 Hz), 0.88 (s, 9H), 0.27 (s,3H), 0.14 (s, 3H).

(e)(1S,11aS)-4-((S)-2-((S)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyl11-(tert-butyldimethylsilyloxy)-8-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(79)

Lithium acetate dihydrate (0.010 g, 0.10 mmol, 1.0 eq) was added to asolution of the silyl ether 78 (0.100 g, 0.10 mmol, 1.0 eq) in wet DMF(2 mL) at 25° C. for 3 hours. The reaction mixture was then diluted withethyl acetate (20 mL) and washed successively with 0.1M citric acid (20mL, pH 3), water (20 mL) and brine (5 mL), dried over MgSO₄, filteredand concentrated under reduced pressure. The residue was purified byflash chromatography (silica gel, 5/95 v/v methanol/DCM) to afford theproduct as a pale yellow oil (0.070 g, 83%). LC/MS (3.362 min (ES⁺)),m/z: 820.2 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (s, 1H), 7.48 (d, 2H,J=8.2 Hz), 7.25 (s, 1H), 7.12 (d, 2H, J=8.1 Hz), 6.88 (s, 1H), 6.68 (s,1H), 6.47 (d, 1H, J=7.6 Hz), 6.24 (d, 1H, J=15.2 Hz), 6.03 (s, 1H), 5.92(m, 1H), 5.84 (d, 1H, J=8.9 Hz), 5.50 (m, 1H), 5.34 (m, 1H), 5.26 (m,2H), 5.18 (d, 1H, J=12.3 Hz), 4.80 (d, 1H, J=12.4 Hz), 4.66-4.60 (m,3H), 4.02 (m, 1H), 3.95 (s, 3H), 3.81 (m, 1H), 3.03 (m, 1H), 2.57 (m,1H), 2.19 (m, 1H), 1.84 (dd, 3H, J=6.8, 0.8 Hz), 1.48 (d, 3H, J=7.1 Hz),1.00 (d, 3H, J=6.8 Hz), 0.95 (d, 3H, J=6.8 Hz), 0.87 (s, 9H), 0.26 (s,3H), 0.12 (s, 3H).

(iv)(11S,11aS)-4-((20S,23S)-1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(66, D)

(a) (11S,11aS)-allyl8-(3-((11S,11aS)-10-((4-((R)-2-((R)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyloxy)carbonyl)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-11-(tert-butyldimethylsilyloxy)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(80)

Potassium carbonate (0.030 g, 0.21 mmol, 1.0 eq) was added to a solutionof the phenol 79 (0.175 g, 0.21 mmol, 1.0 eq) and the iodo linker 74(0.214 g, 0.32 mmol, 1.5 eq) in acetone (10 mL). The reaction mixturewas heated under a nitrogen atmosphere at 75° C. in a sealed flask for17 hours. The reaction mixture was concentrated to dryness under reducedpressure and purified by flash chromatography (silica gel, 2/98 v/vmethanol/DCM to 5/95 v/v methanol/DCM) to afford the product as a paleyellow solid (0.100 g, 35%). LC/MS (4.293 min (ES⁺)), m/z: 1359.13 [M]⁺.

(b) (11S,11aS)-allyl8-(3-((11S,11aS)-10-((4-((R)-2-((R)-2-(allyloxycarbonylamino)-3-methylbutanamido)propanamido)benzyloxy)carbonyl)-11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-11-hydroxy-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(81)

Tetra-n-butylammonium fluoride (1 M, 0.22 mL, 0.22 mmol, 2.0 eq) wasadded to a solution of silyl ether 80 (0.150 g, 0.11 mmol, 1.0 eq) indry THF (2 mL). The reaction mixture was stirred at room temperature for20 minutes, after which LC/MS indicated complete reaction. The reactionmixture was diluted with ethyl acetate (10 mL) and washed sequentiallywith water (5 mL) and brine (5 mL). The organic phase was dried overMgSO₄, filtered and concentrated under reduced pressure to leave ayellow solid. Purification by flash chromatography (silica gel, 6/94 v/vmethanol/DCM to 10/90 v/v methanol/DCM) afforded the product as a paleyellow solid (0.090 g, 73%). LC/MS (2.947 min (ES⁺)), m/z: 1154.0[M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (br s, 1H), 7.39 (d, 2H, J=7.6Hz), 7.18 (d, 2H, J=10.6 Hz), 7.10 (m, 3H), 6.86 (d, 2H, J=10.0 Hz),6.74 (s, 1H), 6.55 (s, 1H), 6.22 (dd, 2H, J=15.3, 6.6 Hz), 5.85 (m, 2H),5.74 (m, 3H), 5.52 (m, 2H), 5.22 (m, 1H), 5.00 (m, 2H), 4.57 (m, 6H),4.41 (m, 2H), 4.09 (m, 4H), 3.85 (m, 11H), 3.06 (m, 2H), 2.76 (m, 2H),2.20 (m, 2H), 2.08 (m, 1H), 1.79 (d, 6H, J=6.4 Hz), 1.40 (d, 3H, J=6.1Hz), 0.90 (m, 6H).

(c)(11S,11aS)-4-((R)-2-((R)-2-amino-3-methylbutanamido)propanamido)benzyl11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(65)

Tetrakis(triphenylphospene)palladium(0) (0.005 g, 0.005 mmol, 0.06 eq)was added to a solution of the bis-carbinolamine 81 (0.090 g, 0.08 mmol,1.0 eq) and pyrrolidine (16 μL, 0.20 mmol, 2.5 eq) in dry DCM (5 mL).After 20 minutes, the reaction mixture was diluted with DCM (10 mL) andwashed sequentially with saturated ammonium chloride (5 mL) and brine (5mL), dried over MgSO₄, filtered and the solvent was removed underreduced pressure to leave the crude product as a pale yellow solid whichwas used in the next step without further purification (0.075 g, assumed100% yield). LC/MS (2.060 min (ES⁺)), m/z: 947.2 [M+H]⁺.

(d)(11S,11aS)-4-((20S,23S)-1-iodo-20-isopropyl-23-methyl-2,18,21-trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl11-hydroxy-7-methoxy-8-(3-((S)-7-methoxy-5-oxo-2-((E)-prop-1-enyl)-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)propoxy)-5-oxo-2-((E)-prop-1-enyl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(66, D)

EDCl (0.015 g, 0.08 mmol, 1.0 eq) was added to a solution of amine 65(assumed 100% yield 0.075 g, 0.08 mmol, 1.0 eq) andiodoacetamide-PEG₄-acid 17 (0.034 g, 0.08 mmol, 1.0 eq) in drydichloromethane (5 mL) and the reaction was stirred in the dark. After50 minutes, a further amount of iodoacetamide-PEG₄-acid 17 (0.007 g,0.016 mmol, 0.2 eq) was added along with a further amount of EDCl (0.003g, 0.016 mmol, 0.2 eq). After a total of 2.5 hours, the reaction mixturewas diluted with dichloromethane (15 mL) and washed sequentially withwater (10 mL) and brine (10 mL). The organic phase was dried over MgSO₄,filtered and concentrated under reduced pressure. The resulting residuewas purified by flash chromatography (silica gel, Chloroform 100% to90:10 v/v Chloroform:Methanol). Pure fractions were combined to affordthe product (0.0254 g, 23%, 2 steps). The crude fractions were collectedand purified by preparative TLC (silica gel, 90:10 v/vChloroform:Methanol) to afford a second batch of product (0.0036 g, 3%,2 steps). LC/MS (2.689 min (ES⁺)), m/z: 681.0 ½[M+2H]⁺.

Example 10: Activity of Released Compounds

K562 Assay

K562 human chronic myeloid leukaemia cells were maintained in RPM1 1640medium supplemented with 10% fetal calf serum and 2 mM glutamine at 37°C. in a humidified atmosphere containing 5% CO₂ and were incubated witha specified dose of drug for 1 hour or 96 hours at 37° C. in the dark.The incubation was terminated by centrifugation (5 min, 300 g) and thecells were washed once with drug-free medium. Following the appropriatedrug treatment, the cells were transferred to 96-well microtiter plates(10⁴ cells per well, 8 wells per sample). Plates were then kept in thedark at 37° C. in a humidified atmosphere containing 5% CO₂. The assayis based on the ability of viable cells to reduce a yellow solubletetrazolium salt,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,Aldrich-Sigma), to an insoluble purple formazan precipitate. Followingincubation of the plates for 4 days (to allow control cells to increasein number by approximately 10 fold), 20 μL of MTT solution (5 mg/mL inphosphate-buffered saline) was added to each well and the plates furtherincubated for 5 h. The plates were then centrifuged for 5 min at 300 gand the bulk of the medium pipetted from the cell pellet leaving 10-20μL per well. DMSO (200 μL) was added to each well and the samplesagitated to ensure complete mixing. The optical density was then read ata wavelength of 550 nm on a Titertek Multiscan ELISA plate reader, and adose-response curve was constructed. For each curve, an IC₅₀ value wasread as the dose required to reduce the final optical density to 50% ofthe control value.

Compound RelC has an IC₅₀ of less than 0.1 pM in this assay.

Compound RelE has an IC₅₀ of 0.425 nM in this assay.

Example 11: Formation of Conjugates

General Antibody Conjugation Procedure

Antibodies are diluted to 1-5 mg/mL in a reduction buffer (examples:phosphate buffered saline PBS, histidine buffer, sodium borate buffer,TRIS buffer). A freshly prepared solution of TCEP(tris(2-carboxyethyl)phosphine hydrochloride) is added to selectivelyreduce cysteine disulfide bridges. The amount of TCEP is proportional tothe target level of reduction, within 1 to 4 molar equivalents perantibody, generating 2 to 8 reactive thiols. After reduction for severalhours at 37° C., the mixture is cooled down to room temperature andexcess drug-linker (A, B, C, D, E) added as a diluted DMSO solution(final DMSO content of up to 10% volume/volume of reaction mixture). Themixture was gently shaken at either 4° C. or room temperature for theappropriate time, generally 1-3 hours. Excess reactive thiols can bereacted with a ‘thiol capping reagent’ like N-ethyl maleimide (NEM) atthe end of the conjugation. Antibody-drug conjugates are concentratedusing centrifugal spin-filters with a molecular weight cut-off of 10 kDaor higher, then purified by tangential flow filtration (TFF) or FastProtein Liquid Chromatography (FPLC). Corresponding antibody-drugconjugates can be determined by analysis by High-Performance LiquidChromatography (HPLC) or Ultra-High-Performance Liquid Chromatography(UHPLC) to assess drug-per-antibody ratio (DAR) using reverse-phasechromatography (RP) or Hydrophobic-Interaction Chromatography (HIC),coupled with UV-Visible, Fluorescence or Mass-Spectrometer detection;aggregate level and monomer purity can be analysed by HPLC or UHPLCusing size-exclusion chromatography coupled with UV-Visible,Fluorescence or Mass-Spectrometer detection. Final conjugateconcentration is determined by a combination of spectroscopic(absorbance at 280, 214 and 330 nm) and biochemical assay (bicinchonicacid assay BCA; Smith, P. K., et al. (1985) Anal. Biochem. 150 (1):76-85; using a known-concentration IgG antibody as reference).Antibody-drug conjugates are generally sterile filtered using 0.2 mfilters under aseptic conditions, and stored at +4° C., −20° C. or −80°C.

DAR Determination

Antibody or ADC (ca. 35 μg in 35 μL) was reduced by addition of 10 μLborate buffer (100 mM, pH 8.4) and 5 μL DTT (0.5 M in water), and heatedat 37° C. for 15 minutes. The sample was diluted with 1 volume ofacetonitrile: water: formic acid (49%: 49%: 2% v/v), and injected onto aWidepore 3.6μ XB-C18 150×2.1 mm (P/N 00F-4482-AN) column (PhenomenexAeris) at 80° C., in a UPLC system (Shimadzu Nexera) with a flow rate of1 ml/min equilibrated in 75% Buffer A (Water, Trifluoroacetic acid (0.1%v/v) (TFA), 25% buffer B (Acetonitrile:water:TFA 90%:10%:0.1% v/v).Bound material was eluted using a gradient from 25% to 55% buffer B in10 min. Peaks of UV absorption at 214 nm were integrated. The followingpeaks were identified for each ADC or antibody: native antibody lightchain (L0), native antibody heavy chain (HO), and each of these chainswith added drug-linkers (labelled L1 for light chain with one drug andH1, H2, H3 for heavy chain with 1, 2 or 3 attached drug-linkers). The UVchromatogram at 330 nm was used for identification of fragmentscontaining drug-linkers (i.e., L1, H1, H2, H3).

A PBD/protein molar ratio was calculated for both light chains and heavychains:

${\frac{Drug}{Protein}\mspace{14mu}{ratio}\mspace{14mu}{on}\mspace{14mu}{light}\mspace{14mu}{chain}} = \frac{{\%\mspace{14mu}{Area}\mspace{14mu}{at}\mspace{14mu} 214\mspace{14mu}{nm}\mspace{14mu}{for}\mspace{14mu} L\; 1}\;}{\%\mspace{14mu}{Area}\mspace{14mu}{at}\mspace{14mu} 214\mspace{14mu}{nm}\mspace{14mu}{for}\mspace{14mu} L\; 0\mspace{14mu}{and}\mspace{14mu} L\; 1}$${\frac{Drug}{Protein}\mspace{14mu}{ratio}\mspace{14mu}{on}\mspace{14mu}{heavy}\mspace{14mu}{chain}} = \frac{\sum\limits_{n = 0}^{g}\;{n\; \times \left( {\%\mspace{14mu}{area}\mspace{14mu}{at}\mspace{14mu} 214\mspace{14mu}{for}\mspace{14mu}{Hn}}\; \right.}}{\sum\limits_{n = 0}^{g}\;{\%\mspace{14mu}{a{rea}}\mspace{14mu}{at}\mspace{14mu} 214\mspace{20mu}{for}\mspace{14mu}{Hn}}}$

Final DAR is calculated as:

${{DAR} = {2 \times \left( {{\frac{Drug}{Protein}\mspace{14mu}{ratio}\mspace{14mu}{on}\mspace{14mu}{light}\mspace{14mu}{chain}} + {\frac{Drug}{Protein}\mspace{14mu}{ratio}\mspace{14mu}{on}\mspace{14mu}{heavy}\mspace{14mu}{chain}}} \right)}}\;$

DAR measurement is carried out at 214 nm because it minimisesinterference from drug-linker absorbance.

Generation of ADCs

Antibody A (comprising a variable domain which is SEQ ID NO. 1 pairedwith SEQ ID NO. 7) was conjugated with drug linker A to give Conj A-A,and the DAR was measured to be 2.35.

Antibody B (comprising a variable domain which is SEQ ID NO. 2 pairedwith SEQ ID NO. 8) was conjugated with drug linker A to give Conj B-A,and the DAR was measured to be 2.2 (see table below).

Antibody C (comprising a variable domain which is SEQ ID NO. 6 pairedwith SEQ ID NO. 12) was conjugated with drug linker A to give Conj C-A,and the DAR was measured to be 1.97.

Antibody D (comprising a variable domain which is SEQ ID NO. 4 pairedwith SEQ ID NO. 10) was conjugated with drug linker A to give Conj D-A,and the DAR was measured to be 1.99.

Antibody E (comprising a variable domain which is SEQ ID NO. 5 pairedwith SEQ ID NO. 11) was conjugated with drug linker A to give Conj E-A,and the DAR was measured to be 1.89.

Antibody B (comprising a variable domain which is SEQ ID NO. 2 pairedwith SEQ ID NO. 8) was conjugated with drug linker D to give Conj B-D,and the DAR was measured to be 2.18 (see table below).

Antibody B (comprising a variable domain which is SEQ ID NO. 2 pairedwith SEQ ID NO. 8) was conjugated with drug linker E to give Conj B-E,and the DAR was measured to be 1.93 (see table below).

RB4v1.2 is an anti-CD19 antibody comprising a VH domain having thesequence according to SEQ ID NO. 2 and a VK domain having the sequenceaccording to SEQ ID NO. 8.

As described above, ADCs targeted to CD19 were generated by conjugatingRB4v1.2 antibodies to the drug linker A, E, or D. The resulting ADCs arelisted in the table below, alongside the measured DAR. B12 anti-HIVgp120 antibody was used to generate control non-CD19 targeted ADCs.

ADC DAR Concentration [mg/ml] Yield [%] RB4v1.2-A 2.2 0.683 56 RB4v1.2-E1.93 0.636 66 RB4v1.2-D 2.18 0.859 57 B12-A 2.03 0.98 71 B12-E 1.840.515 70 RB4v1.2-DM4 3.27 2.62 81

Example 12: In Vitro Cytotoxicity of ADCs

Cell Culture

WSU-DLCL2 and SU-DHL-1 cells were from the Leibniz Institute DSMZ-GermanCollection of Microorganisms and Cell Cultures. Ramos and Daudi cellswere from the American Type Culture Collection. Cell culture medium wasRPMI 1640 supplemented with L-Glutamine and 10% FBS. Cells were grown at37° C., 5% CO₂, in a humidified incubator.

Cytotoxicity Assay

The concentration and viability of cultures of suspended cells (at up to1×10⁶/ml) were determined by mixing 1:1 with Trypan blue and countingclear (live)/blue (dead) cells with a haemocytometer. The cellsuspension was diluted to the required seeding density (generally10⁵/ml) and dispensed into 96-well flat bottomed plates. For Alamar blueassay, 100 μl/well was dispensed in black-well plates. For MTS assay, 50μl/well was dispensed in clear-well plates. A stock solution (1 ml) ofADC (20 μg/ml) was made by dilution of filter-sterile ADC into cellculture medium. A set of 8×10-fold dilutions of stock ADC were made in a24 well plate by serial transfer of 100 μl onto 900 μl of cell culturemedium. Each ADC dilution (100 μl/well for Alamar blue, 50 μl/well forMTS) was dispensed into 4 replicate wells of the 96-well plate,containing cell suspension. Control wells received the same volume ofculture medium only. After incubation for 4 days, cell viability wasmeasured by either Alamar blue or MTS assay.

AlamarBlue® (Invitrogen, catalogue number DAL1025) was dispensed (20 μlper well) into each well and incubated for 4 hours at 37° C. in theCO₂-gassed incubator. Well fluorescence was measured at excitation 570nm, emission 585 nm. Cell survival (%) was calculated from the ratio ofmean fluorescence in the 4 ADC-treated wells compared to the meanfluorescence in the 4 control wells (100%).

MTS (Promega, catalogue number G5421) was dispensed (20 μl per well)into each well and incubated for 4 hours at 37° C. in the CO₂-gassedincubator. Absorbance was measured at 490 nm. Cell survival (%) wascalculated from the mean absorbance in the 4 ADC-treated wells comparedto the mean absorbance in the 4 control wells (100%). Dose responsecurves were generated from the mean data of 3 replicate experiments andthe EC₅₀ was determined by fitting data to a sigmoidal dose-responsecurve with variable slope using Prism (GraphPad, San Diego, Calif.).

In Vitro Cytotoxicity

The efficacy of RB4v1.2-A was tested against CD19 +ve cell lines, Daudi,Ramos and WSU-DLCL2. As a CD19 −ve control, SU-DHL-1 cells were used.

FIG. 1 shows in vitro efficacy of RB4v1.2-A. Serial 10-fold dilutions(μg/mL) of RB4v1.2-A were incubated with Daudi, Ramos, WSU-DLCL2 orSU-DHL-1 cells. The Alamar Blue assay was performed at the end of theincubation period and cell survival calculated. Graphs represent theaverage of three replicate experiments.

RB4v1.2-A showed significant cytotoxicity against Daudi and Ramos cell,with slightly lower cytotoxicity against WSU-DLCL2 cells (FIG. 1).

RB4v1.2-A SUDHL1 Daudi Ramos WSU-DLCL2 EC50 (μg/mL) ~6.706 0.0011150.001021 0.1171

FIG. 2 shows in vitro efficacy of RB4v1.2-E. Serial 10-fold dilutions(μg/mL) of RB4v1.2-E were incubated with Daudi, Ramos, WSU-DLCL2 orSU-DHL-1 cells. The Alamar Blue assay was performed at the end of theincubation period and cell survival calculated. Graphs represent theaverage of three replicate experiments.

RB4v1.2-E showed significant cytotoxicity against Daudi and Ramos cell,with slightly lower cytotoxicity against WSU-DLCL2 cells (FIG. 2).

RB4v1.2-E SUDHL1 Daudi Ramos WSU-DLCL2 EC50 (μg/mL) 0.5390 0.0012160.001467 0.03099

FIG. 3 shows in vitro efficacy of RB4v1.2-D. Serial 10-fold dilutions(μg/mL) of RB4v1.2-D were incubated with Daudi, Ramos, WSU-DLCL2 orSU-DHL-1 cells. The Alamar Blue assay was performed at the end of theincubation period and cell survival calculated. Graphs represent theaverage of three replicate experiments.

RB4v1.2-D showed significant cytotoxicity against Daudi and Ramos cell,with slightly lower cytotoxicity against WSU-DLCL2 cells (FIG. 3).

RB4v1.2-D SUDHL1 Daudi Ramos WSU-DLCL2 EC50 (μg/mL) ~0.7540 0.0010220.0007979 0.02190

Example 13—In Vivo Anti-Tumour Activity of ADCs

The CD19+(ve) human Burkitt's lymphoma-derived cell line Ramos was usedto determine the most potent PBD drug linker for RB4v1.2 in vivo. Theantibody was conjugated to A, E, or D drug linkers as described aboveand tested in the Ramos xenograft model. As non-CD19-binding controls,the anti-HIV gp120 antibody, B12, linked to A or E was used. At the sametime, to investigate any potential anti-tumor activity observed with theRB4v1.2 without a PBD warhead, RB4v1.2 was tested in vivo conjugated toDM4, a maytansinoid derivative which acts as a tubulin inhibitor.

Study Design

Drugs and Treatment:

Animals Dose Dose Group per level volume No group ADC (mg/kg) (ml/kg) 110 [no treatment] — — 2 10 [vehicle only] — — 3 10 RB4v1.2 [no PBD] 1.010 4 10 RB4v1.2-A 0.3 10 5 10 RB4v1.2-A 1.0 10 6 10 RB4v1.2-E 0.3 10 710 RB4v1.2-E 1.0 10 8 10 RB4v1.2-D 0.3 10 9 10 RB4v1.2-D 1.0 10 10 10B12-E 1.0 10 11 10 B12-A 1.0 10Procedures:

-   -   Set up CR female NCr nu/nu mice with I x Ramos-SPN tumor cells        in 0% Matrigel sc in flank.    -   Cell Injection Volume is 0.1 mL/mouse.    -   Age at Start Date: 8 to 12 weeks.    -   Perform a pair match when tumors reach an average size of        100-150 mm³ and begin treatment.    -   Body Weight: qd×5 then bi-wk to end    -   Caliper Measurement: bi-wk to end    -   Report any adverse reactions or death immediately.    -   Any individual animal with a single observation of >30% body        weight loss or three consecutive measurements of >25% body        weight loss will be euthanized.    -   Any group with two measurements of mean body weight loss of >20%        or >10% mortality will stop dosing. The group is not euthanized        and recovery is allowed. Within a group with >20% weight loss,        individuals hitting the individual body weight loss endpoint        will be euthanized. If the group treatment related body weight        loss is recovered to within 10% of the original weights, dosing        may resume at a lower dose or less frequent dosing schedule.        Exceptions to non-treatment body weight % recovery may be        allowed on a case-by-case basis.    -   Endpoint TGD. Animals are to be monitored individually. The        endpoint of the experiment is a tumor volume of 2000 mm³ or 60        days, whichever comes first. Responders can be followed longer.        When the endpoint is reached, the animals are to be euthanized.        General Methodological Approach

For the calculation of group mean tumor volumes the following rule wasapplied: when an animal exited the study due to tumor size, the finaltumor volume recorded for the animal was included with the data used tocalculate the mean volume at subsequent time points. Error bars indicatestandard error of the mean (SEM). Tumor volumes values were not used tocalculate group mean tumor volumes when fewer than 50% of the animals ina group remained in the study. Prism (GraphPad, San Diego, Calif.) wasused for graphical presentations and statistical analyses.

Results

FIG. 4 shows drug linker selection for RB4v1.2-ADCs. Mice were dosedwhen the mean tumor volume per experimental group reached 0.1 cm³ andthey were treated with a single dose of the ADCs at 0.3 and 1 mg/kg (forbinding ADCs) and 1 mg/kg (for non-binding-ADCs) via IV in the tailvein. Data represent the mean tumour volume (+/−SEM) from ten mice ineach group.

RB4v1.2-A, -E and -D all exhibited anti-tumor activity, with RB4v1.2-Eand -D appearing more potent than RB4v1.2-A (see FIG. 4). Neithernon-binding ADC controls (B12-A and B12-E) showed a significantanti-tumor activity.

ABBREVIATIONS

-   Ac acetyl-   Acm acetamidomethyl-   Alloc allyloxycarbonyl-   Boc di-tert-butyl dicarbonate-   t-Bu tert-butyl-   Bzl benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is    methylbenzene-   Cbz or Z benzyloxy-carbonyl, where Z—Cl and Z—Br are chloro- and    bromobenzyloxy carbonyl respectively-   DMF N,N-dimethylformamide-   Dnp dinitrophenyl-   DTT dithiothreitol-   Fmoc 9H-fluoren-9-ylmethoxycarbonyl-   imp N-10 imine protecting group:    3-(2-methoxyethoxyl)propanoate-Val-Ala-PAB-   MC-OSu maleimidocaproyl-O—N-succinimide-   Moc methoxycarbonyl-   MP maleimidopropanamide-   Mtr 4-methoxy-2,3,6-trimethtylbenzenesulfonyl-   PAB para-aminobenzyloxycarbonyl-   PEG ethyleneoxy-   PNZ p-nitrobenzyl carbamate-   Psec 2-(phenylsulfonyl)ethoxycarbonyl-   TBDMS tert-butyldimethylsilyl-   TBDPS tert-butyldiphenylsilyl-   Teoc 2-(trimethylsilyl)ethoxycarbonyl-   Tos tosyl-   Troc 2,2,2-trichlorethoxycarbonyl chloride-   Trt trityl-   Xan xanthyl

SEQUENCES SEQ ID NO. 1 (RB4v1.0 VH):QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGS NPYYYAMDYWGQGTSVTVSSEQ ID NO. 2 (RB4v1.2 VH):QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQAPGQGLEWIGEIDPSDSYTNYNQNFQGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGS NPYYYAMDYWGQGTSVTVSSEQ ID NO. 3 (B43 VH):QVQLLESGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLRSEDSAVYSCARRE TTTVGRYYYAMDYWGQGTTVTSEQ ID NO. 4 (HD37 VH):QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVS SEQ ID NO. 5 (4G7 VH):EVQLQQSGPELIKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGT YYYGSRVFDYWGQGTTLTVSSEQ ID NO. 6 (FMC63 VH):EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSEQ ID NO.7 (RB4v1.0 VK):EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTK LEIKSEQ ID NO. 8 (RB4v1.2 VK):EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTK LEIKSEQ ID NO. 9 (B43 VK):ELVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPW TFGGGTKLEIKSEQ ID NO. 10 (HD37 VK):DILLTQTPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWT FGGGTKLEIKSEQ ID NO. 11 (4G7 VK):DIVMTQAAPSIPVTPGESVSISCRSSKSLLNSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYP FTFGAGTKLELKSEQ ID NO. 12 (FMC63 VK):DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEIT

The invention claimed is:
 1. A conjugate of formula

where Ab is an antibody which is an antibody that binds to CD19, theantibody comprising a VH domain having the sequence of SEQ ID NO. 2,optionally further comprising a VL domain having the sequence of SEQ IDNO. 8; and wherein the drug loading is from 1 to
 8. 2. The conjugateaccording to claim 1 wherein the antibody comprises a VH domain pairedwith a VL domain, the VH and VL domains having sequences of: SEQ ID NO.2 paired with SEQ ID NO.
 8. 3. The conjugate according to claim 1wherein the antibody is an intact antibody.
 4. The conjugate accordingto claim 1 wherein the antibody is humanised, deimmunised or resurfaced.5. The conjugate according to claim 1, wherein the drug loading is 1, 2,3, or
 4. 6. A mixture of the conjugates according to claim 1, whereinthe average value of the drug loading is about 2 to about
 5. 7. Theconjugate according to claim 1, wherein said conjugate comprises one ofmore conjugates selected from the group consisting of ConjA, ConjD andConjE for use in treating a CD19 overexpressing cancer.
 8. The conjugateaccording to claim 7, wherein the conjugate is ConjE.
 9. Apharmaceutical composition comprising the conjugate of claim 1 and apharmaceutically acceptable diluent, carrier or excipient.
 10. Thepharmaceutical composition of claim 9 further comprising atherapeutically effective amount of a chemotherapeutic agent.
 11. Amethod of treating a CD19 overexpressing cancer comprising administeringto a patient the pharmaceutical composition of claim 9 wherein saidconjugate comprises one of more conjugates selected from the groupconsisting of ConjA, ConjD and ConjE.
 12. The method of claim 11 whereinthe patient is administered a chemotherapeutic agent, in combinationwith the conjugate.
 13. A method of claim 11 wherein said conjugate isConjE.
 14. The method according to claim 12, wherein thechemotherapeutic agent is rituximab.
 15. The method according to claim13, wherein the chemotherapeutic agent is rituximab.
 16. The conjugateaccording to claim 2, wherein the conjugate is formula ConjE.